Novel methods for the in-vitro identification, isolation and differentiation of vasculogenic progenitor cells

ABSTRACT

There is provided a simplified and inexpensive method for the in-vitro identification, isolation and culture of human vasculogenic progenitor cells. The method and the progenitor cells isolated thereby can be used for in-vitro vascular engineering, treatment of congenital and acquired vascular and hematological abnormalities, for evaluation and development of drugs affecting vasculo- and angiogenic processes, and for further investigation into tissue differentiation and development.

[0001] This application claims the benefit of priority from U.S.Provisional Patent Application No. 60/372,429, filed Apr. 16, 2002.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to novel methods for the isolationand culture of vasculogenic progenitor cells from stem cells and, moreparticularly, to methods for use of vasculogenic progenitor cells intissue engineering, research and diagnostics.

[0003] Recently, techniques have been developed which allow humanembryonic stem cells to proliferate indefinitely in culture, enablingexperimentation with induction of differentiation in a directed,tissue-specific manner (Itskovitz-Eldor, J et al Mol Med 2000;6:88-95,Reubinoff B E at al Nat Biotech 2000;18:399-404, Schuldiner M et al PNASUSA 2000;97:11307-12). Human embryonic stem cell growth and developmentis being carefully studied, and the rapidly accumulating knowledge isbeing employed in a variety of innovative therapeutic applicationsincluding in-vitro tissue engineering, transplantation medicine,generation of transgenic embryos and treatment of degenerative disease.Most significantly, the President of the U.S. has recognized theoverwhelming importance of embryonic stem cells to medicine andresearch, and has recently sanctioned projects using existing humanembryonic stem cell lines (White House Fact Sheet: Embryonic Stem CellResearch, Aug. 9, 2001). However, in-vitro manipulation of the complexsteps of development, to reliably produce substantial amounts of desiredcell lineages and specific phenotypes remains a crucially importantgoal.

[0004] Blood Vessel Formation in Embryonic Development and Adult Life

[0005] In the early stages of embryonic development, vessel formationoccurs by a process referred to as vasculogenesis, in whichmesodermally-derived endothelial cell progenitors undergo de-novodifferentiation, expand and coalescence to form a network of primitivetubules (Yancopoulos G D et al Nature 2000;407:242). These blood vesselsare generally composed of two cell lineages, each serving a differentfunction: internal endothelial cells that form the channels for bloodconduction, but alone cannot complete vasculogenesis; andperiendothelial smooth muscle cells that protect and stabilize thefragile channels from rupture and provide haemostatic control (CarmelietP Nature Med 2000;6:389). A third cell lineage, the hematopoietic cells,share a common progenitor with the vascular cells, and differentiateinto the blood cells. In the vertebrate embryo vasculogenesis occurs inthe paraxial and lateral mesoderm, giving rise to the primordia of theheart, the dorsal aorta, and large vessels of the head, lung andgastrointestinal system. Angiogenesis involves the maturation andremodeling of the primitive vascular plexus into a complex network oflarge and small vessels. Angiogenesis also leads to vascularization ofinitially avascular organs such as kidney, brain and limb buds.

[0006] Angiogenesis is also required postnatally for normal tissuegrowth, and continues throughout adult life, for example duringneo-vascularization of the endometrium during normal female estrus,during pregnancy in the placenta, and during wound healing (Risau, et alNature 1997; 386:671-674).

[0007] In addition, a number of diseases and disorders have beenassociated with abnormal endothelial growth: endothelialhyperproliferation in atherosclerosis, neovascularization in tumorgrowth and metastasis, and deregulated angiogenesis in rheumatoidarthritis, retinopathies, hemangiomas and psoriasis (Folkman et alNature Med. 1995;1: 27-31; Hanahan and Folkman, Cell 1996;86:353-64).

[0008] Embryonic Endothelial Cells In-Vitro

[0009] Research into the functions, origin and nature of embryonicendothelial cells (EEC) has revealed that EECs can promote liverorganogenesis (Matsumoto K et al Science 2001;294:559), induce pancreasdifferentiation (Lammert E et al 2001;294:564) and trans-differentiateinto cardiac muscle cells under specific conditions (Condorelli G et al2001;98:10733). While the nature of differentiation and development ofendothelial precursors is not yet fully understood, it is becoming clearthat hematopoietic development and the generation of vascular smoothmuscle cells (v-SMC) are tightly linked with vascular development.

[0010] Embryonic stem cells are difficult to maintain in culture,tending to spontaneously differentiate. For ongoing cultures, cells fromthe inner mass of blastocysts are typically grown on a layer of mouseembryonic fibroblast “feeder” cells to preserve their undifferentiatedphenotype and proliferabilty (Keller, G M Curr Opin Cell Biol1995;7:862-69). In mice, early differentiation into embryonicallydistinct cell types can be induced by coculture with stromal cell lines(Palacios R, et al PNAS USA 1995;92:7530-34), culture on substrates suchas fibronectin, laminin, collagen, etc. (Ogawa M et al Blood 1999;93:1168-77) or in vitro aggregation of embryoid stem (ES) cells into“embryoid bodies” (EB), demonstrating regional differentiation intothree germ layers (Keller, GM Curr Opin Cell Biol 1995;7:862-69).

[0011] Murine Embryonic Stem Cells

[0012] Study of vasculogenic events in murine ES cells has beeninstructive. Both hematopoietic and endothelial cells have been observedin blast cell colonies generated from mouse ES cell-derived embryoidbodies (Choi K, et al Development 1998;125:725). Also working withmurine ES cells, Nishikawa and colleagues demonstrated that 3-D embryoidbody formation was not required for differentiation of lateral mesodermcells. When cultured non-aggregated mouse embryonic cells were grown ona collagen substrate, cells expressing vascular endothelial Cadherin(VE-cad+) were found to give rise to hematopoietic cells (Nishikawa S I,et al Development 1998;125:1747, Nishikawa S I et al Immunity1998;8:761, and Fujimoto T, et al Genes Cells 2001;6:1113). Wheremarkers of smooth muscle cell (SMC) phenotype (e.g. surface markers andmorphology) are observed, early periendothelial SMCs associated withembryonic endothelial tubes can be shown to trans-differentiate from theendothelium (Gittenberger de-Groot, A C et al, Atheroscler Thromb VascBiol 1999;19:1589), and differentiation of embryonic common vascularprogenitors (Flk1+) into endothelial and smooth muscle cells can beenobserved (Yamashita J et al Nature 2000; 408:92). However, attempts todirectly extrapolate from mouse to human EC systems have met withdisappointing results, indicating that many developmental processes andrequirements are species specific (see, for example, Reubinoff B E etal, Nat. Biotechnolog. 2000;18:399-404). Specifically, in contrast toit's expression in mouse embryonic stem (mES) cells, the vascularspecific growth factor receptor VEGFR 2 (Flk-1/KDR) is expressed inundifferentiated hES cells (Kaufman, D S et al PNAS USA2001;98:10716-21) and does not increase during the first week ofdifferentiation (Levenberg, S et al PNAS USA 2002;99:4391-96),indicating that the timing of VEGFR 2 expression may vary amongvertebrate species (also reviewed by Nishikawa; Nishikawa S I et al CurrOpin Cell Biol 2001;13:862-69). Levenberg et al (Levenberg, S et al PNASUSA 2002;99:4391-96) further reported that other endothelial markers,namely vascular endothelial cadherin (VE-cad) and platelet-endothelialcell adhesion molecule-1 (PECAM1/CD31), increased during the first weekof hES differentiation. Clearly, coordination of expression of specificendothelial-specific factors, in the appropriate combinations, arecrucial to human vasculogenesis.

[0013] Human Embryonic Stem Cells

[0014] Human embryonic stem (hES) cell lines were first derived in 1998(Thomson, J A et al Science 1998;282:1145; U.S. Pat. No. 6,200,806 toThomson et al; U.S. Pat. No. 6,331,406 to Gearhart J D and Shamblott MJ), and have recently been induced to differentiate in vitro in a celllineage-specific manner (Schuldiner M et al PNAS 2000;97:11307-312,International Patent Application WO0210347 A2 to Benvenisty, N). SincehES cells maintain the embryonic stem cell phenotype throughout hundredsof doubling times, and differentiate to all embryonic cell lineages,they provide a potentially unlimited source of cells for study andclinical application. Both hematopoietic and endothelial celldifferentiation have been observed in human ES cells. To date,hematopoietic differentiation of the hES cells has required coculturingwith either the S17 (murine bone marrow) or C166 (yolk sac endothelial)stromal cell lines, inducing the appearance of primary humanhematopoietic tissue characteristics such as cell surface antigen CD34and hematopoietic colony formation (Kaufman, DS et al PNAS USA2001;98:10716-21). In another recent study, endothelial cells wereselected by cell sorting (FACS) from human embryoid bodies (EB) usingmonoclonal antibodies raised against the endothelial-specific markerPECAM-1 (Levenberg, S et al PNAS USA 2002;99:4391-96). The selected,PECAM-1+ embryoid body-derived (EBD) cells exhibitedendothelial-specific characteristics such as von Willebrand factor,VEGFR-2 and VE-cad surface markers and primitive, vessel-like cordformation when cultured on a soft substrate (Matrigel). PECAM-1+ EBDcells were further observed forming vascular structures in-vivofollowing seeding on biodegradable polymer matrix sponges andimplantation into SCID mice. However, all of the abovementioned methodsfor differentiation of human ES require either coculturing withnon-human cells or embryoid body formation prior to appearance ofendothelial phenotypes, and immunofluorescent cell sorting for selectionaccording to endothelial cell markers, rendering them both costly andunsuitable for many clinical applications. Thus, it would beadvantageous to provide a simplified, less expensive method ofculturing, selecting and directing differentiation of human embryonicstem cells, without the limitations of aggregation into embryoid bodiesor immunofluorescent selection.

[0015] Prior art discloses a number of techniques and methods forpreparation and use of embryonic stem cells for differentiation. Earlytechniques required inner-cell mass cells from blastocyst-stage embryos(fresh or cryopreserved) as a source of stem cells (see, for example,International Patent Application No. WO 0129206 A1 to Cibelli et al;U.S. Pat. Applications Nos. 20020045259 A1 to Lim et al, 20020004240 A1to Wang). Many others rely upon aggregation of the stem cells intoembryoid bodies for initiation of differentiation (see, for example,International Patent Application No. WO0070021 A3 to Itskovitz-Eldor Jand Benvenisty N).

[0016] Various methods for differentiation of stem cells in culture havealso been disclosed. International Patent Application No. WO 0134776 A1,U.S. Pat. Application No. 20020015694 A1, and U.S. Pat. No. 6,280,718,all to Kaufman, D et al, disclose methods of differentiating humanembryonic stem cells into hematopoietic cells by coculture withmammalian stromal cells. U.S. Patent Application No. 20020023277 A1 toStuhlmann, H et al discloses the identification and isolation of thevasculogenesis-related gene Vezf1 in mice, and methods for selection ofendothelial cells and precursors based on Vezf1 expression. Alsodisclosed are methods for modulating angiogenesis, and diagnosis andtreatment of vascular disease and neoplasm in a subject, the methodsemploying detection, measurement and modification of levels of Vezf1 intissues. However, the transgenic ES cell experiments described wererestricted to mouse embryoid body cells only, and neither human nor anyother primate embryo cells were used. Furthermore, selection, accordingto the disclosure, is on the basis of Vezf1 expression, thus failing toovercome the abovementioned limitations of aggregation andimmunofluorescent sorting.

[0017] U.S. Patent Application No. 20020039724 A1 to Carpenter, M Kdiscloses methods for differentiation and selection of human embryonicneural progenitor cells, and therapeutic, diagnostic and investigativeuses thereof. The disclosed human neural progenitor cells, forreconstitutive therapy of, for example, neural degenerative disease, arealso derived from human embryoid bodies, and are selected and isolatedaccording to expression and detection of neural cell specific markers,NCAM and A2B5. Similarly, International Patent Application WO 0181549 A3to Rambhatla L and Carpenter MK discloses methods for treating embryoidbodies with n-butyrate for induction of differentiation into hepatocytelineage cells. No mention is made of non-aggregated hES origins, orsimplified methods of progenitor isolation in either application.

[0018] Recently, Benevenisty (International Patent Application WO0210347 A2 to Benvenisty) disclosed methods for “directeddifferentiation” of human embryonic stem cells by treating aggregated,embryoid body-derived cells with exogenous factors, enriching thecultures for a specific lineage cell type. The factors used were knowneffectors of differentiation, such as retinoic acid, neuronal growthfactor, epidermal growth factor, fibroblast growth factor, etc., anddifferentiation was determined by de novo gene expression, and theappearance of tissue lineage-specific cell surface markers.

[0019] U.S. Pat. Application No. 20010041668 A1, to Baron, M et al,discloses the use of extraembryonic, morphogenic gene products such asHedgehog, TNF and WNT for modulation of hematopoiesis and vasculargrowth from mammalian adult and embryonic mesodermal-derived stem cells.Manipulation of the levels of these extra-embryonic gene products in thestem cell environment, via external application, or genetic engineering,for example, is disclosed for either enriching or diminishing thehematopoietic and/or vascular potential of stem cells for treatment anddiagnosis of diseases involving blood abnormalities,hypervascularization, neovascularization and revascularization oftissues. However, although treatment of human embryonic tissues isproposed, no examples using human adult or embryonic cells arepresented, and no methods for culture or selection of non-aggregatedembryonic stem cells, designed to overcome the abovementionedlimitations, are disclosed.

[0020] Thus, there exists a need for a simplified and inexpensive methodfor the in-vitro identification, isolation and culture of humanvasculogenic progenitor cells. Such a method and the progenitor cellsisolated thereby can be used for in-vitro vascular engineering,treatment of congenital and acquired vascular and hematologicalabnormalities, for evaluation and development of drugs affectingvasculo- and angiogenic processes, and for further investigation intotissue differentiation and development.

SUMMARY OF THE INVENTION

[0021] According to one aspect of the present invention there isprovided a method of preparing vasculogenic progenitor cells fromundifferentiated ES cells, the method effected by culturing individualundifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells, thereby obtaining a mixed population of cells; andisolating cells smaller than 50 μm from said mixed population of cells,said cells smaller than 50 μm being vasculogenic progenitor cells.

[0022] According to another aspect of the present invention there isprovided a method of preparing epithelial progenitor cells fromundifferentiated ES cells, the method effected by culturing individualundifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells thereby obtaining a mixed population of cells; andisolating cells larger than 50 μm from said mixed population of cells,said cells larger than 50 μm being epithelial progenitor cells.

[0023] According to yet another aspect of the present invention there isprovided a method of preparing somatic cells from a population ofvasculogenic progenitor cells, the method effected by obtaining apopulation of vasculogenic progenitor cells; and culturing thepopulation of vasculogenic progenitor cells in the presence of at leastone growth factor suitable for inducing somatic cell differentiation.

[0024] According to still another aspect of the present invention thereis provided a method of preparing vascular tissue, the method effectedby obtaining a population of vasculogenic progenitor cells; andculturing the population of vasculogenic progenitor cells in thepresence of at least one vasculogenic and/or angiogenic growth factor,under conditions suitable for inducing vascular tissue differentiation.

[0025] According to further features in preferred embodiments of theinvention described below the population of vasculogenic progenitorcells is cultured in a semi-solid, vascularization-promoting medium.

[0026] According to yet further features in preferred embodiments of theinvention described below the population of vasculogenic progenitor iscultured on a 3-dimensional scaffold.

[0027] According to still further features in preferred embodiments ofthe invention described below the vasculogenic and/or angiogenic factoris selected from the group consisting of vascular endothelial growthfactor (VEGF), angiopoietin (Ang), platelet derived growth factor(PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growthfactor (TGF) and placental growth factor (PIGF).

[0028] According to an additional aspect of the present invention thereis provided a method of determining an effect of a factor on vasculardevelopment, growth and/or modification, the method effected byobtaining a population of vasculogenic progenitor cells; exposing thepopulation of vasculogenic progenitor cells to the factor; anddetermining an effect of the factor on the population of vasculogenicprogenitor cells to thereby determine the effect thereof on vasculardevelopment.

[0029] According to further features in preferred embodiments of theinvention described below the factor is a substance and/or anenvironmental factor.

[0030] According to yet further features in preferred embodiments of theinvention described below the factor is a putative angiogenesis and/orvasculogenesis downregulator, whereas the method further includesculturing the population of vasculogenic progenitor cells underconditions suitable for promoting angiogenesis and/or vasculogenesis.

[0031] According to still further features in preferred embodiments ofthe invention described below the factor is a putative angiogenesisand/or vasculogenesis upregulator, whereas the method further includesculturing the population of vasculogenic progenitor cells underconditions limiting angiogenesis and/or vasculogenesis.

[0032] According to a further aspect of the present invention there isprovided a method of relieving or preventing a vascular disease orcondition in a mammalian subject, the method effected by obtaining apopulation of vasculogenic progenitor cells; and administering thevasculogenic progenitor cells into the subject under conditions suitablefor stimulating differentiation of the vasculogenic progenitor cellsinto endothelial and smooth muscle cells.

[0033] According to further features in preferred embodiments of theinvention described below the vascular disease or condition is selectedfrom a group consisting of congenital vascular disorders, acquiredvascular disorders and ischemia/reperfusion injury.

[0034] According to yet a further aspect of the present invention thereis provided a method of vascularizing a mammalian tissue, the methodeffected by obtaining a population of vasculogenic progenitor cellscontacting the vasculogenic progenitor cells with the mammalian tissueunder conditions suitable for stimulating differentiation of thevasculogenic progenitor cells into endothelial and smooth muscle cells.

[0035] According to further features in preferred embodiments of theinvention described below the mammalian tissue is an engineered,non-vascular tissue in need of vascularization and/or an embryonictissue.

[0036] According to further features in preferred embodiments of theinvention described below contacting the vasculogenic progenitor cellswith the mammalian tissue is performed in vitro or in vivo.

[0037] According to still a further aspect of the present inventionthere is provided a method of relieving or preventing a hematologicaldisease or condition in a mammalian subject, the method effected byobtaining a population of vasculogenic progenitor cells; andadministering the vasculogenic progenitor cells into the subject underconditions suitable for stimulating differentiation of the vasculogenicprogenitor cells into endothelial and blood cells.

[0038] According to further features in preferred embodiments of theinvention described below the hematological disease or condition isselected from a group consisting of congenital blood disorders, acquiredblood disorders, clotting disorders and neoplastic disease.

[0039] According to further features in preferred embodiments of theinvention described below obtaining the population of vasculogenic cellsis effected by culturing individual undifferentiated ES cells in amanner suitable for inducing differentiation of the undifferentiated EScells into vasculogenic progenitor cells thereby obtaining a mixedpopulation of cells and isolating cells smaller than 50 μm from saidmixed population of cells.

[0040] According to still an additional aspect of the present inventionthere is provided a composition of matter comprising a substrate and apopulation of vasculogenic progenitor cells, wherein said vasculogenicprogenitor cells are prepared from undifferentiated ES cells by a methodeffected by the steps: culturing individual undifferentiated ES cells ina manner suitable for inducing differentiation of the undifferentiatedES cells into vasculogenic progenitor cells thereby obtaining a mixedpopulation of cells and isolating cells smaller than 50 μm from saidmixed population of cells, said cells smaller than 50 μm beingvasculogenic progenitor cells.

[0041] According to further features in preferred embodiments of theinvention described below the substrate is selected from the groupconsisting of matrigel, collagen gel, and polymeric scaffold.

[0042] According to still further features in preferred embodiments ofthe invention described below the vasculogenic progenitor cells iscontacted with the substrate in a manner so as to induce vasculardevelopment within the substrate.

[0043] According to further features in preferred embodiments of theinvention described below the hematological disease or condition isselected from a group consisting of congenital blood disorders, acquiredblood disorders, clotting disorders and neoplastic disease.

[0044] According to yet further features in preferred embodiments of theinvention described below culturing the individual undifferentiated EScells is effected by subjecting the undifferentiated ES cells to atleast one condition selected from a group consisting of avoidingaggregation of ES cells, growth on collagen, cell seeding concentrationbetween 2×10⁴ and 1×05 cells/cm² and presence of differentiation medium.

[0045] According to still further features in preferred embodiments ofthe invention described below the undifferentiated ES cells are human EScells.

[0046] According to an additional aspect of the present invention thereis provided a method of preparing endothelial cells from vasculartissue, the method effected by subjecting the vascular tissue toconditions designed for dissociating cells from the vascular tissue,thereby obtaining a mixed population of dissociated cells and isolatingcells smaller than 50 μm from said mixed population of cells.

[0047] According to a further aspect of the present invention there isprovided a method of preparing epithelial cells from vascular tissue,the method effected by subjecting the vascular tissue to conditionsdesigned for dissociating cells from the vascular tissue, therebyobtaining a mixed population of dissociated cells, thereby obtaining amixed population of individual cells; and isolating cells larger than 50μm from said mixed population of cells.

[0048] According to further features in preferred embodiments of theinvention described below the vascular tissue is human vascular tissueAccording to yet further features in preferred embodiments of theinvention described below the cells smaller or larger than 50 μm areisolated via filtration, morphometry and/or densitometry.

[0049] According to still further features in preferred embodiments ofthe invention described below the filtration is effected via a filterhaving a pore size smaller than 50 μm.

[0050] According to yet an additional aspect of the present inventionthere is provided a cell culture comprising a population of vasculogenicprogenitor cells being sustainable in a proliferative state for at least14 days and being capable of differentiation into smooth muscle,endothelial and/or hematopoietic cells upon exposure to at least onegrowth factor selected from the group consisting of vascular endothelialgrowth factor (VEGF), angiopoietin (Ang), platelet derived growth factor(PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growthfactor (TGF), placental growth factor (PIGF), cytokines, erythropoietin,thrombopoietin, transferrin, insulin, stem cell factor (SCF),Granulocyte colony-stimulating factor (G-CSF) and Granulocyte-macrophagecolony stimulating factor (GM-CSF).

[0051] According to further features in preferred embodiments of theinvention described below the population of vasculogenic progenitorcells is capable of expressing at least one exogenous polypeptideselected from the group consisting of cell-surface markers, cell-surfaceantigens, angiogenic factors, vasculogenic factors and hematopoieticfactors.

[0052] According to still further features in preferred embodiments ofthe invention described below the exogenous polypeptide is expressed inan inducible manner.

[0053] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

[0055] In the drawings:

[0056] FIGS. 1A-H provide an outline for, and micrographs demonstratingthe culture-based selection for human ES cell-derived vasculogenicprogenitor cells. FIG. 1A illustrates the outline of thedifferentiation-selection procedure. FIG. 1B is a series of micrographsdemonstrating the divergent morphology of the cells following 6 daysculture on collagen: note the large, flat fiber-bearing cells (arrows)and the smaller, flattened cells with large nuclei (arrowheads). FIG. 1Cis a series of graphs demonstrating a FACS analysis of endothelial cellsurface markers in the filtered, isolated vasculogenic progenitorscells. Filtered cells were exposed to primary antibodies to VE-cadhedrin(VE-cad), VEGFR2 (VEGFR2), and to fluorescent labeled anti-IgG, or tothe second antibody alone (IgG-FITC). Note the high proportion of cells(78%) expressing VE-cad. FIGS. 1D-E are photographs demonstrating theindirect immunomorphological analysis of VE-cad expression on filtered,isolated vasculogenic progenitors cells. Immunofluorescent staining offixed and plated 12 hour cultures of the filtered cells demonstratestrong localization of VE-cad, at cell-cell adherent junctions, visibleat higher magnification (FIG. 1E). FIG. 1F is a photograph of EtBrstained gels demonstrating the expression of endothelial andhematopoietic markers in the isolated vasculogenic progenitors cells.Expression of the CD31 and Tie2 endothelial markers and the Tall, GATA2and AC 133 early vasculogenic progenitor markers was compared in totalRNA from the smaller, flat filtered cells (Filtrated), and theundifferentiated human embryonic stem (hES) cells by RT-PCR. Thehousekeeping marker GAPDH serves as an internal standard ofamplification. Note the prominent, endothelial, smooth muscle andhematopoietic (ESH)-specific expression of the CD31, Tie2, Tall andGATA2 markers. FIG. 1G is a fluorescent micrograph of the larger, flatcells retained by filtration, demonstrating the presence of theepitheliod phenotype smooth muscle cell marker α-sma not detected in thesmaller, human vasculogenic progenitor cells. FIG. 1H is a photograph ofEtBr stained gels demonstrating the expression of epitheliod markers inthe isolated, larger retained cells. Expression of the Calponin,Caldesmon, smooth muscle actin (SMA) and SM-MHC markers was compared intotal RNA from the larger, flat retained cells (Retained), and thesmaller, human vasculogenic progenitor (Filtrated) cells by RT-PCR. Thehousekeeping marker GAPDH serves as an internal standard ofamplification. Note the absence of expression of all of the smoothmuscle cell markers in the human vasculogenic progenitor cells, andtheir prominent expression in the Retained cells. FIG. 1I is amicrograph of BrdU incorporation in both smaller, filtered (left panel),and larger, retained cells (right panel), demonstrating activeproliferation of the smaller, human vasculogenic progenitor cells. Notethe active uptake of BrdU in the smaller, darkened cell nuclei,contrasted with the minimal incorporation in the larger,non-proliferating retained cells (arrow). Bar equals 10 μm.

[0057] FIGS. 2A-M are microscopic, immunofluorescence and RT-PCR studiesof cultured common human vasculogenic progenitor cells, demonstratingspecific growth factor-mediated induction of endothelial or smoothmuscle cell characteristics. FIG. 2A is a photograph of EtBr stainedgels demonstrating the expression of smooth muscle cell markers incommon human vasculogenic progenitor cells recultured on type IVcollagen at 2.5×10⁴ cells/cm² for 10-12 days with 10 ng/ml hPDGF-BB (Rand D Systems, Inc., Minneapolis, Minn., USA). Expression of the smoothmuscle cell markers Caldesmon, smooth muscle actin (SMA), Calponin,SM22α and SM-MHC markers was compared in total RNA from the growthfactor-treated cells (v-SMC), and the untreated human vasculogenicprogenitor (ESH progenitor) cells by RT-PCR. The housekeeping markerGAPDH serves as an internal standard of amplification. Note the absenceof expression of all of the smooth muscle cell markers in the ESH cells,and their prominent expression in the hPDGF-BB treated cells. FIGS. 2B-Eare photomicrographs of immunofluorescent detection of smooth musclecell markers expressed in the human platelet-derived growth factor(hPDGF)-BB-treated human vasculogenic progenitor cells. Fixedpreparations of treated cells were stained with primary antibodies to: αSMA (FIG. 2B); smoothelin (FIG. 2C); SM-MHC (FIG. 2D), and Calponin(FIG. 2E), immunodetected with fluorescent second antibodies andvisualized via fluorescent microscopy. Note the staining of bothepitheliod and spindle-shaped cell types in the growth factor-treatedcultures. FIGS. 2F-H are photomicrographs showing the detection ofendothelial cell markers expressed in human vasculogenic progenitorcells recultured on type IV collagen at 2.5×10⁴ cells/cm² for 10-12 dayswith 50 ng/ml hVEGF₁₆₅ (R and D Systems, Inc. Minneapolis, Minn., USA).Growth factor-treated cells were fixed and immune-detected as describedhereinabove with anti-VEcad (FIG. 2F), or anti-von Willebrand Factor(vWF)(FIG. 2G) antibodies. Note the localization of anti vWF staining inthe Weibel-Palade bodies (FIG. 2G). Uptake of Dill-labeled ac-LDL (10μg/ml, 4 hours, 37° C.)(FIG. 2h) was also detected (FIG. 2H). FIGS. 2Iand 2J are micrographs of BrdU incorporation in both platelet derived(PDGF) (FIG. 2J) and vascular endothelium (VEGF) (FIG. 2I) growthfactor-treated human vasculogenic progenitor cells, demonstrating activeproliferation (staining) of the endothelial-type cells. Note theappearance of stress fibers (FIG. 2I, arrow), and the active uptake ofBrdU in the darkened cell nuclei of the VEGF-treated cells (FIG. 2I),contrasted with the reduced incorporation of the larger hPDGF-BB treatedcells (FIG. 2J). FIGS. 2K-2M are micrographs of hematopoietic coloniesformed from human vasculogenic progenitor cells. ESH cells wereselected, and cultured in a semisolid medium supplemented with cytokinesto promote hematopoietic differentiation. Note the characteristicappearance of hematopoietic colonies (CFU) detected after 12 daysincubation.

[0058] FIGS. 3A-G are photomicrographs (FIGS. 3A-D), and electronmicrographs (FIGS. 3E-G) showing vascular structure formation ingrowth-factor-treated human vasculogenic progenitor (ESH) cells.Aggregated (24 hours in differentiation medium supplemented with 50ng/ml hVEGF₁₆₅ and 10 ng/ml hPDGF-BB) ESH cells seeded onto type Icollagen (FIG. 3A) or in matrigel (FIG. 3B) demonstrated vascularformation after 7 days growth (Note sprouting and tubular structures inboth histology sections). Toluidine blue-stained sections of the samepreparation revealed endothelial cell penetration and formation of avascular-network structure in the matrigel (FIG. 3C) and, with highermagnification, a white blood cell formed within a vessel (arrow, FIG.3D). Bar equals 10 μm (FIGS. 3A-C) or 20 μm (FIG. 3D). FIGS. 3E-G areelectron micrographs of vessel formation in Matrigel, showingwell-formed Weibel-Palade bodies (FIG. 3E, ×6,000 magnification, inset,×12,000), typical of endothelial cells. FIG. 3F clearly demonstrates thepresence of a darkly staining (due to Hemoglobin) blood cell (BC) in thecenter of a vessel formed by elongated endothelial cells (EC) within thematrigel (M) (×5,000 magnification). FIG. 3G demonstrates typicalarrangement of endothelial cells (N-nucleus) within the matrigel (M),containing a clearly discernible lumen (Lu), characteristic lipoproteincapsules (Li), Weibel-Palade bodies (WP) and glycogen (G) (FIG. 3G,×5,000 magnification).

[0059] FIGS. 4A-B are photomicrographs of histology sections depictingthe in vitro vascularization of 3-D alginate scaffolds by humanvasculogenic progenitor (ESH) cells. ESH aggregates were seeded on 1f12050 μl alginate scaffolds in vitro in differentiation medium supplementedwith 50 ng/ml hVEGF₁₆₅ and 10 ng/ml hPDGF-BB, and incubated for 14 days.FIG. 4A shows vessel formation around two representative scaffold pores.Higher magnification (FIG. 4B) reveals typical vascular wall structureof elongated flat endothelial cells with an adjacent layer of smoothmuscle cells. Bar equals 10 μm.

[0060] FIGS. 5A-B are two series of photomicrographs demonstrating thesensitivity of ESH-derived vascular tissue to inhibitors ofangiogenesis. ESH aggregates were seeded on matrigel and incubated for 7days in differentiation medium supplemented with 50 ng/ml hVEGF₁₆₅ and10 ng/ml hPDGF-BB alone (FIG. 5A) or with the addition of 50 μg/mlangiogenesis inhibiting anti VE-cad monoclonal antibody (clone BV6,CHEMICON INTNL, Inc. Temecula Calif., USA)(FIG. 5B). Note the lack ofcellular projections and absence of tube and network structures in theanti VE-cad treated cultures (FIG. 5B). Bar equals 10 μm.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention is of novel methods which can be used forsimple and inexpensive preparation of vasculogenic progenitor cells, andcell cultures and compositions thereof prepared from, for example, humanstem cells. Specifically, the present invention can be used forisolating vasculogenic progenitor cells from stem cells, and for invitro growth and differentiation of the isolated vasculogenic progenitorcells for use in, for example, tissue engineering, angiogenesisresearch, therapeutic and diagnostic applications.

[0062] The principles and operation of the present invention may bebetter understood with reference to the drawings and accompanyingdescriptions. Before explaining at least one embodiment of the inventionin detail, it is to be understood that the invention is not limited inits application to the details of construction and the arrangement ofthe components set forth in the following description or illustrated inthe drawings. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

[0063] Recent research studies have illustrated that embryonic cells canpotentially serves as a source for pluripotent cells. Such cells areuseful in human therapy since they posses the capacity to differentiateinto a plurality of cell types (R. A. Pedersen, Scientif. Am.1999;280:68). Early work on embryonic stem cells was done using inbredmouse strains as a model. Compared with mouse ES cells, monkey and humanpluripotent cells have proven to be much more fragile, and do notrespond to the same culture conditions and manipulations.

[0064] Recently, human embryonic stem cells (hES) and germ-line (hEG)cells have been isolated and maintained in culture. Both human embryonichES and hEG cells have the long-sought characteristics of humanpluripotent stem cells, they are capable of ongoing proliferation invitro without differentiating, they retain a normal karyotype, and theyretain the capacity to differentiate to produce all adult cell types.However, spontaneous somatic differentiation of hES and hEG cells inculture proceeds without any consistent pattern of structuralorganization, generating multicellular aggregates of cell populationswith a highly heterogeneous mixture of phenotypes, representing aspectrum of different cell lineages (Reubinoff, B E, et al Nat Biotech2001; 19:1134).

[0065] Prior art studies describe various methods suitable for isolationof progenitor cells of specific cell type lineages from ES cells,however, such methods are typically extremely complex and costly.Initially, human embryonic stem cells are either grown on a mammalianstromal cell layer (see, for example, U.S. Pat. No. 6,280,718 toKaufman, D S and Thomson, J A), in a live host as a teratoma (Thomson JA et al Science 1998;282:1145-47) or aggregated in suspension into amulticellular structure known as the embryoid body (EB) (see, forexample, International Pat. Application No. WO0070021 A3 toItskovitz-Eldor J and Benvenisty N; and International Pat. ApplicationWO0210347 A2 to Benvenisty N), and exposed to differentiation factors,typically producing a mixed population of cell types and lineages.Isolation of progenitor cells of specific lineages is then accomplishedon the basis of immunodetection of lineage-specific markers, andseparation of cell lineages by fluorescent or magnetic sorting (see, forexample, International Pat. Application No. W 00181549 A3 to Rambhatla Land Carpenter, MK; U.S. Pat. No. 6,280,718 to Kaufman, D S and Thomson,J A; International Pat. Application No. WO0129206 A1 to Cibelli, J etal; and International Pat. Application No. WO 0168815 A1 to Pera, M Fand Ben-Hur T). All of the abovementioned methods suffer from similardisadvantages: initial ES differentiation into progenitor cells involvesmany complex manipulations and interactions with the stromal cell layer,live host tissues or other EB cells. Furthermore, selection according toexpression or display of cell surface markers is inefficient, requiringeven more extensive manipulation, incurring great expense for reagentsand detection equipment, and endangering the vitality and sterility ofthe progenitor cells.

[0066] While reducing the present invention to practice the presentinventors have uncovered that vasculogenic progenitor cells may beprepared simply and inexpensively from embryonic stem cells bypreventing aggregation, culturing on type IV collagen with specificendothelial differentiation factors and employing simple and efficientsize selection methods. The vasculogenic progenitor cells prepared bythe present invention are advantageous in that they can be furtherexpanded in culture, can be induced to differentiate into endothelial,mural and hematopoietic tissue in vitro, form both small and largevascular structures when seeded on appropriate substrate, may begenetically manipulated and are suitable for tissue engineering,diagnostic and research purposes.

[0067] Thus, according to one aspect of the present invention there isprovided a method of preparing vasculogenic progenitor cells fromundifferentiated ES cells, such as human ES cells. The method, accordingto this aspect of the present invention, is effected by culturingindividual undifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells, thereby obtaining a mixed population of cells, andisolating cells smaller than 50 μm from the mixed population of cells.Cell isolated in this manner are vasculogenic progenitor cells as isclearly illustrated in the Examples section hereinunder.

[0068] As used herein, the phrase “vasculogenic progenitor cells” refersto a population of cells that can generate progeny that are endothelialor smooth muscle precursors (such as angioblasts) or mature endothelialor smooth muscle cells, or hematopoietic precursor (such as erythroidcolony forming units and megakaryocytes) or mature blood cells (such aserythrocytes and leukocytes). Typically, vasculogenic progenitor cellsexpress some of the phenotypic markers that are characteristic of theendothelial, smooth muscle and hematopoietic lineages. Typically, theydo not produce progeny of other embryonic germ layers when cultured bythemselves in vitro, unless dedifferentiated or reprogrammed. It will beappreciated that it is not implied that each of the cells within thepopulation have the capacity of forming more than one type of progeny,although individual cells that are multipotent vasculogenic progenitorcells may be present.

[0069] As used herein the, the terms “totipotent”, “pluripotent” and“multipotent” refer to cells having decreasing degrees of developmentalplasticity. Totipotent cells are capable of developing into all celltypes or complete organisms (e.g. blastomeres), pluripotent cellscapable of differentiating into all cell types (e.g. ES cells) andmultipotent cells are capable of differentiating into cells of specificlineages only (e.g. vasculogenic progenitor cells).

[0070] As used herein, the term “endothelial progenitor cell” or“endothelial precursor cell” refers to a cell that can generate matureendothelial cells. These cells may or may not have the capacity togenerate hematopoietic or smooth muscle cells.

[0071] As used herein, the term “epithelial progenitor cell” or“epithelial precursor cell” refers to a cell that can generate maturesmooth muscle cells.

[0072] As used herein, the term “hematopoietic progenitor cell” or“hematopoietic precursor cell” refers to a cell that can generate matureblood cells.

[0073] Embryonic stem cells are described as “undifferentiated” when asubstantial portion of stem cells and their derivatives in thepopulation display morphological characteristics of undifferentiatedcells, clearly distinguishing them from differentiated cells ofembryonic or adult origin. Undifferentiated ES cells are easilyrecognized by those skilled in the art, and typically appear in amicroscopic view as cells with high nuclear/cytoplasm ratios andprominent nucleoli. Similarly, undifferentiated cells can bedistinguished from differentiated cells by the absence of lineagespecific markers such as vascular endothelial growth factor receptor 2(VEGFR2), vascular endothelial cadherin (VE-cad) or platelet-endothelialcell adhesion molecule-1 (PECAM-1).

[0074] As used herein, the term “differentiated cell” refers to a cellthat has progressed down a developmental pathway. Thus, pluripotentembryonic stem cells can differentiate to lineage-restricted precursorcells, such as neural progenitor, hepatocyte progenitor or hematopoieticcells, which are pluripotent for neural cells, hepatocytes and bloodcell types, respectively; and the endothelial, smooth muscle and bloodcell types listed above. These in turn may be differentiated furtherinto other types of precursors further down the pathways, or to anend-stage differentiated cell, which is characteristic of a specifictissue type, and may or may not retain the capacity to proliferatefurther. Vascular endothelium, mural smooth muscle and erythrocytes areexamples of terminally differentiated cells.

[0075] As mentioned hereinabove, individual undifferentiated ES cellsare cultured in a manner suitable for inducing differentiation intovasculogenic progenitor cells. The undifferentiated ES cells utilized bythe method of the present invention can be mammalian embryonic stemcells obtained from fresh or cryopreserved embryonic cell masses, cellsfrom in-vitro-fertilized embryonic cell masses and/or cultured ES celllines. The ES cells may be of human or non-human origin.

[0076] As is clearly demonstrated in the Examples section hereinbelow,the methods and compositions of the present invention are suitable foruse with human embryonic stem cells. Since establishment of methods formanipulation and control of human embryonic stem cell differentiation isa primary goal of current medical and scientific effort, in a preferredembodiment of the present invention, the undifferentiated ES cells arehuman ES cells. Preferably, the ES cells are unaggregated cells, asdescribed in detail in the Examples section hereinbelow.

[0077] According to another preferred embodiment of the presentinvention, differentiation of the individual undifferentiated ES cellsis effected by culturing such cells on plates coated with an adhesivesubstrate such as type IV collagen, laminin or gelatin to preventaggregation of the ES cells, seeding the cells at a concentrationbetween 2×10⁴ and 1×10⁵ cells/cm², and providing differentiation medium.In a most preferred embodiment, individual undifferentiated ES cells aregrown on type IV collagen-coated plates (available from, for example,Cell Cultureware, BD-Falcon, Boston, Mass.). See Examples section forfurther description of conditions for differentiation of ES cells.

[0078] One important feature of the present methodology is the cellseeding step. While reducing the present invention to practice, it wasobserved that a 3-dimensional embryoid body structure was not required,as had been previously contended, for mesodermal differentiation ofhuman embryonic stem cells. Undifferentiated hES cells removed fromtheir feeder layer and plated as single cells on type IV collagen withdifferentiation medium exhibited expression of indicators of endothelialdifferentiation (FIGS. 1A-G). Cell seeding concentration dramaticallyaffected the efficiency of the present methodology: cells seededaccording to prior art studies with mouse ES (Yamashita, J et al Nature2000;408:92) were not viable; such high concentrations (1.0-1.5×10⁵cells/cm²) of hES cells resulted in a heterogeneous population. Lowercell seeding concentrations (5×10⁴-1×10⁵ cells/cm²) produced a definedpopulation of cells, including a majority of small, flatendothelial-like cells and fewer large, smooth-muscle-like cells (FIG.1B).

[0079] The Examples section which follows provides further descriptionof methods of culturing “individual undifferentiated ES cells” undernon-aggregating conditions.

[0080] As used herein, the term “differentiation medium” refers to asuitable medium capable of supporting growth and differentiation of theES cells. Examples of suitable differentiation media which can be usedwith the present invention include a variety of growth media preparedwith a base of alpha MEM medium (Life Technologies Inc., Rockville, Md.,USA) or Dulbecco's minimal essential medium (DMEM) supplemented with 10%FBS (HyClone, Logan, Utah, USA) and 0.1 mM β-mercapoethanol (LifeTechnologies Inc., Rockville, Md., USA).

[0081] As is mentioned hereinabove it was observed that culturing of theundifferentiated ES cells as detailed hereinabove produces a definedpopulation of cells, including a majority of small, flatendothelial-like cells and fewer large, smooth-muscle-like cells (FIG.1B).

[0082] While previous techniques for selection of specific lineageprogenitors have depended on immunodetection of indicators ofdifferentiation and specific cell lineages and fluorescent or magneticcell sorting (see, for example, International Patent Application WO0210347 A2 to Benvenisty, U.S. Pat. No. 6,280,718 to Kaufman, D S andThomson, J A; International Pat. Application No. WO0129206 A1 toCibelli, J et al), these methods are cumbersome and costly. The observedmorphological features of the mixed population of cells generatedaccording to the teachings of the present invention enabled a simple andrapid isolation of vasculogenic progenitor cells therefrom. As isillustrated in the Examples section which follows, selection of cellssmaller than 50 μm, enables rapid and efficient isolation ofvasculogenic progenitor cells from the mixed population of cells (FIGS.1C-F).

[0083] Thus, the present methodology employs a step of size/morphologyselection following differentiation. Such size/morphology selection canbe effected using various filtration, morphometry and/or densitometryapproaches as is further described below.

[0084] Methods of filtration are well known in the art, such as thepassage through a mesh, sieve, filter and the like. Filters can comprisea fibrous matrix or porous material. Such filters may be one of severalcommercially available filters including but not limited to cell culturefilters from Pall Life Sciences (Ann-Arbor Mich., USA) or BD-Falcon(Boston, Mass., USA). A preferred filter is a nylon mesh filter having apore size of 40 μm (Cell Cultureware, BD-Falcon, Boston, Mass.),allowing the smaller, endothelial-like cells to pass and the larger,smooth-muscle like cells to be excluded.

[0085] “Morphometry” refers to the measurement of external form, and canemploy methods including but not limited to 2- and 3-D image analysis.Advanced imaging analysis software suitable for identification andisolation of cells smaller than 50 μm is commercially available to oneskilled in the art [see, for example, Metamorph Software (UniversalImaging Corp., Downing Pa., USA), Imagic-5 (Image Science Software,Berlin, Germany) and Stereologer (Systems Planning and Analysis, Inc.,Alexandria, Va., USA)] and can be combined with well known lightmicroscopy and flow sorting techniques for selection of objects ofdesired external characteristics (e.g. size) (for suitable apparatussee, for example, U.S. Pat. No. 6,249,341 to Basiji et al).“Densitometry” refers to measurement of the optical or physical densityof an object. Since the smaller, endothelial-like cells have a uniqueand characteristic distribution of cell components, densitometricmeasurements may be used to characterize and provide criteria forseparation and isolation of cells. Devices suitable for densitometricisolation of endothelial-like cells are, for example, the MECOS-C1 bloodcell densitometry analyzer (MECOS Co., Moscow, Russia). Cells may alsobe separated by sedimentation through a preparative density gradientsuch as FICOLL™ or PERCOLL™ (Amersham Biosciences, Inc. Piscataway, N.J.USA) (for exhaustive review of densitometric fractionation techniques,see Pertoft, H J Biochem Biophys Methods 2000; 44:1-30). Thus, thepresent invention provides an easy and rapid approach to progenitor cellgeneration and isolation. Previous methods of isolating such progenitorcells have produced progenitor populations which lack desirableproliferation capabilities, limiting their practical application(Reubinoff, B E et al Nat Biotech 2000;18:399-404, and Schuldiner, M etal PNAS USA 2000;97:11307-312). The vasculogenic progenitor cellsisolated by the methods of the present invention are capable ofgenerating large numbers of identical cells by proliferation throughnumerous cell doublings.

[0086] The population of vasculogenic progenitor cells isolatedaccording to the teachings of the present invention is characterized byan abundance of cells expressing the endothelial progenitor markerVE-cadhedrin (FIGS. 1C-E) and endothelial markers (FIG. 1F), andactively proliferating, as indicated by incorporation of (BrdU) into thenucleus (FIG. 1I). In the absence of additional stimulus for furtherdifferentiation, these cells are capable of generating large numbers ofmultipotent vasculogenic progenitor cells. In addition, the vasculogenicprogenitor cells may be maintained in a viable state over exceedinglylong periods of time by cryopreservation according to any of the methodsfor conditioning, storage and thawing typically employed in the art(see, for example, U.S. Pat. No. 6,140,123 to Demetriou, et al).

[0087] Due to the importance of differentiated cells in varioustherapeutic approaches, directed differentiation of embryonic precursorcells presents an important goal in the art of stem cell culturing.Although embryonic stem cells maintained in culture often undergospontaneous differentiation (Thomson J. A. et al Science1998;282:1145-47), directed differentiation of embryoid body-derivedcells (Shamblott M J et al PNAS USA 2001;98:113-18) and human ES cellsin coculture with MEF cells (Kaufman DS et al PNAS USA2001;98:10716-721) has been demonstrated by manipulation ofenvironmental factors. For example, Kaufman et al induced hematopoieticdifferentiation in human ES cells by culture with mouse bone marrowstromal cells (Kaufman D S et al PNAS USA 2001;98:10716-721, and U.S.Pat. No. 6,280,718 to Kaufman, D et al) and Carpenter (US PatApplication No. 20020039724 A1) induced neuronal and glial celldevelopment in neural progenitor cells by exposure to a cAMP activatorand/or neurotrophic growth factor. Benvenisty produced pulsating cardiacmuscle cells and neuron-like cells by exposing human embryoid body cellsto a variety of growth factors (Itskovitz-Eldor, J et al Mol Med2000;6:88-95, Schuldiner, M et al PNAS USA 2000;97:11307-312 andInternational Pat Application No. WO 0210347 A2 to Benvenisty N).However, all of the abovementioned methods employ either coculturing,embryoid body formation or selection of progenitors by immunodetectionof cell-surface markers.

[0088] Directed differentiation of the vasculogenic progenitor cells ofthe present invention can be effected by exposure to specific vascular,smooth muscle or hematopoietic growth factors.

[0089] As is illustrated in the Examples section which follows, exposureof the vasculogenic progenitor cells seeded at a low concentration, togrowth factors, induces differentiation into specific mature cellphenotypes. Exposure to the growth factor hVEGF induced the appearanceof both morphological and functional indicators of endothelial cellphenotype (FIGS. 1C-F and 2F-H), while exposure to the smooth musclegrowth factor hPDGF-BB upregulated smooth muscle cell markers (FIGS.2A-E). Similarly, exposure to cytokines stimulated hematopoieticdifferentiation of the vasculogenic progenitor cells (FIGS. 2K-M).

[0090] Thus, according to another aspect of the present invention thereis provided a method of preparing somatic cells from the population ofvasculogenic progenitor cells of the present invention, the method iseffected by obtaining a population of vasculogenic progenitor cells asdescribed hereinabove, and culturing the population of vasculogenicprogenitor cells in the presence of at least one growth factor suitablefor inducing somatic cell differentiation.

[0091] As used herein, the term “somatic cell” refers to a cell ofdefinite lineage, identifiable as belonging to a specific cell phenotypevia morphological, immunological, biochemical and/or functionalcriteria. Somatic cells are by definition more differentiated, and lessmultipotent, than progenitor and stem cells. Examples of somatic cells,in the context of the present invention, are endothelial cells, smoothmuscle cells, and blood cells.

[0092] Numerous growth factors have been implicated in the complexprocesses of vasculogenesis, angiogenesis and hematopoieticdifferentiation (for reviews, see Carmeliet, P Nature Med 2000;6:389-95,and Yancopoulos G Nature 2000;407:242-48). Although some (i.e. VEGF, Angand PDGF) are more dominant in their effects than others, effectivedifferentiation of progenitor cells into somatic cells is typically aresult of the combined, and temporally coordinated action of a number offactors.

[0093] Thus, according to one embodiment of this aspect of the presentinvention, directed differentiation is effected by using one or moregrowth factors including, but not limited to, vascular endothelialgrowth factor (VEGF), angiopoietin (Ang), platelet derived growth factor(PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growthfactor (TGF), placental growth factor (PIGF), cytokines, erythropoietin,thrombopoietin, transferrin, insulin, stem cell factor (SCF),Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophagecolony stimulating factor (GM-CSF). Such factors are commerciallyavailable to one skilled in the art, in preparations suitable for use incell culture.

[0094] Furthermore, it will be appreciated that the abovementionedgrowth factors may comprise families of factors including relatedmolecules having different, and divergent roles in the developmentalprocess. Thus, exposure to members of the VEGF family (for exampleVEGF-A, VEGF-B . . . VEGF-D), GM-CSF and bFGF may stimulate endothelialdifferentiation, while the PDGF and Ang families are important in smoothmuscle development and lumen formation, respectively.

[0095] While reducing the present invention to practice, it was revealedthat following initial exposure to differentiation medium and sizeselection by filtration, the vasculogenic progenitor cells, and not thesmooth muscle progenitors of the present invention demonstrate robustnuclear uptake of BrdU, indicating cell proliferation.

[0096] Thus, according to another aspect of the present invention, thereis provided a cell culture comprising a population of vasculogenicprogenitor cells being sustainable in a proliferative, undifferentiatedstate for as long as 14 days or more and being capable ofdifferentiation into smooth muscle, endothelial and/or hematopoieticcells upon exposure to at least one angiogenic, vasculogenic orhematopoietic growth factor, as detailed hereinabove. Thus, the cellculture of the present invention can be expanded and maintained in arelatively undifferentiated state.

[0097] The pluripotent, and proliferative character of embryonic andadult stem cells has naturally been exploited for the benefit of invitro tissue preparation and engineering. In tissue engineering, tissueprogenitors or precursors are cultured in vitro with appropriatedifferentiation factors, to achieve not only differentiation on thelevel of the individual cells, but also morphological, biochemical andanatomical organization into recognizable and functional tissue andorgan structures, which may be used as a source for tissue/organ grafts,for artificial organ support, or organ-bioreactors. Examples of tissuesthat have been engineered in vitro are cartilage (Koch R J and Gorti G KFacial Plast Surg 2002;18:59-68), skin (Lee K H Yonsei Med J2000;41:774-79), genitourinary tissues (Atala A Curr Opin Urol1999;9:517-26) and pancreatic islets (Maria-Engler S S et al Braz J MedBiol Res 2001;34:691-7). However, these tissues have been engineeredfrom differentiated tissue components, not from stem cells. Embryonicstem cells have been used to produce functional pancreatic islet-likestructures (Lemelsky, et al Science 2001;292:1389-94) and blood tissue(Kaufman D S et al PNAS USA 2001;98:10716-21) in vitro.

[0098] Vessel-like structures have also been formed in vitro. Kaushal etal (Nat Med 2001;7:1035-40) reported peripheral endothelial progenitorsforming functional neovessels on decellularized porcine vessels.Levenberg et al (PNAS USA 2002;99:4391-96), working with human embryoidbody derived endothelial cells, demonstrated formation of tube-likestructures in matrigel, and microvessels upon transplantation. However,these vessel-like structures typically lack the normal complexvascular/mural organization characteristic of normal blood vessels.

[0099] While reducing the present invention to practice, it wassurprisingly uncovered that the vasculogenic progenitor cells of thepresent invention form small, capillary-like vessels when grown inmatrigel with appropriate growth factors (FIGS. 3A-G), and largervascular structures on alginate scaffolds (FIGS. 4A and 4B). In bothcases, normal endothelial and mural organization were observed (FIGS.3E-G and 4A-B), as well as blood cell formation within the vascularstructures.

[0100] Thus, according to another aspect of the present invention, thereis provided a method for preparing vascular tissue. The method iseffected by culturing the population of vasculogenic progenitor cells ofthe present invention in the presence of at least one vasculogenicand/or angiogenic growth factor, under conditions suitable for inducingvascular tissue differentiation.

[0101] According to one embodiment of this aspect of the presentinvention, vascular tissue is prepared by culturing the vasculogenicprogenitor cells in a semi-solid, vascularization-promoting medium. Sucha medium typically comprises extracellular matrix components (forexample, Matrigel- B D Biosciences, Bedford, Mass. USA) or collagen(e.g. rat tail collagen I), in which growth factor-treated,differentiating vasculogenic cells are mixed following aggregation. Thegrowth factors may be any of the abovementioned vasculogenic and/orangiogenic factors, such as vascular endothelial growth factor (VEGF),angiopoietin (Ang), platelet derived growth factor (PDGF), ephrin (Eph),fibroblast growth factor (FGF), tumor growth factor (TGF) and placentalgrowth factor (PlGF), known to induce vasculogenic and/or angiogenicgrowth or development. In a preferred embodiment, the growth factors are50 ng/ml VEGF₁₆₅ and 10 ng/ml hPDGF-BB. Growth of vascular structures istypically evident after 7-15 days incubation. Characteristic endothelialcell components, such as Weibel-Palade bodies and lipoprotein capsules;vessel lumen, and blood cells are detected by histology and electronmicroscopy, as detailed in the Examples section which follows. Complexmacroscopic tissue architecture may also be mimicked in vitro by seedingthe progenitors of the present invention on a porous support, orscaffold. Such supports are well known in the art (see U.S. Pat. Nos.5,759,830 and 5,770,417 to Vacanti et al, and 6,379,962 to Holy et al),and have been recently proposed, for example, as tubular blood vesselprostheses for vascularization and epithelialization by host cells, forvascular regeneration (U.S. Pat Application 20020019663 A1 to Termin, PLet al), for wound repair with fibroblasts (U.S. Pat Application20020076816 to Dai, J et al) and for in vitro bone engineering (U.S. PatApplication No. 20020028511 to deBruijn, JD et al). In one embodiment ofthe present invention, vascular tissue of greater than capillary size isprepared by culturing the vasculogenic progenitor cells on a3-dimensional scaffold. In a preferred embodiment, the scaffold is aporous, biodegradable sponge-like material such as poly-L lactic acid,polylactic-glycolic acid or alginate, and differentiation mediumcontains growth factors VEGF₁₆₅ (50 ng/ml) and hPDGF-BB (10 ng/ml).Vascular tissue of the present invention grown on such an alginatescaffold typically demonstrates vascular characteristics such as lumen,endothelial and smooth muscle cells, cell inclusions and von WillebrandFactor at 14 days in culture (FIGS. 4A-B).

[0102] Living vascular tissue prepared by the method of the presentinvention can be used for regenerative therapy, and forneovascularization of non-vascular tissue. Vascular tissue may beimplanted into embryonic, growing or adult organisms suffering frominsufficient or faulty vascularization, as in the microvascularpathology of diabetes, or into tissues experiencing, or at risk ofischemic damage, as in ischemic heart disease and cerebral-vasculardisease. Similarly, vascular tissue of the present invention can provideblood vessels of large diameter for tissue replacement therapy in casesof surgical bypass, vascular degeneration such as atherosclerosis andautoimmune disease.

[0103] It will be appreciated that differentiating cultures or vasculartissues prepared from vasculogenic progenitor cells of the presentinvention also provide a model suitable for the investigation ofprocesses effecting vascular development and function. For example, thecells and tissues of the present invention may be cultured in thepresence of suspected toxic materials, antibodies, teratogens, drugs andthe like, or exposed to non-standard environmental factors such astemperature, gas partial pressure and pH, or co-cultured in the presenceof cells from other tissues or other organisms. Changes in parameters ofgrowth and development, such as failure or delay of endothelial markerexpression, loss of proliferative capacity, or dis-organization of invitro vascularization can be assessed to determine the effect of variousfactors.

[0104] Thus, according to another aspect of the present invention, thereis provided a method of determining an effect of a factor on vasculardevelopment, growth and/or modification. The method is effected byexposing the population of vasculogenic progenitor cells of the presentinvention to the factor, and determining an effect of the factor on thecells.

[0105] The vasculogenic progenitor cells can be exposed to a factorsuspected of inhibiting or downregulating vascular development, growthor modification. Such assays are well known in drug development andresearch, and may be employed to test undesirable side effects ofsubstances intended for the treatment of other, non-vascular processes,or, alternatively, may be used to discover novel inhibitors ofvasculogenesis. In order to enable assessment of effects inhibitingvascular development and growth, conditions of culturing thevasculogenic progenitor cells should be favorable, or more preferably,optimal, for vasculogenesis and angiogenesis. This includes optimizationof medium components (such as growth or differentiation factors),temperature, substrate composition, gas partial pressures and the like,for the specific stage of vascular development being investigated.

[0106] Indeed, while reducing the present invention to practice, thepresent inventors found that incubation of vasculogenic progenitor cellsof the present invention with an angiogenesis-inhibiting anti VE-cad mAbprevented differentiation by hVEGF (FIGS. 5A-B). Similarly, a drugintended for treatment of early complications of pregnancy could bescreened for potential harmful effects on embryonic vasculardevelopment, by exposing vasculogenic progenitor cells, removing thedrug and monitoring modulation of growth or development of the cells bymethods commonly used in the art. Similarly, factors stimulating orupregulating angiogenesis and/or vasculogenesis in the vasculogenicprogenitor cells can be best assessed under sub-optimal conditions ofculturing. Substances affecting vasculogenesis and/or angiogenesisinclude peptides, peptidomimetics, polypeptides, antibodies, chemicalcompounds and biological agents.

[0107] Since progenitor cell populations are highly amenable to tissueengineering, transplantation and regenerative therapy, geneticmanipulation of such cells can provide a source of developing cellpopulations bearing unique, previously unattainable characteristics.

[0108] As is clearly illustrated in Example 1 of the Examples sectionhereinbelow, the vasculogenic progenitor cell population of the presentinvention exhibits active proliferation thus making such cells amenableto genetic manipulations rendering such cells, for example, capable ofexpressing at least one exogenous polypeptide. Exogenous polypeptidesexpressed in such a cell culture can be cell surface markers,cell-surface antigens, angiogenic factors, vasculogenic factors andhematopoietic factors. Additional polypeptides that can be expressedare, for example, various receptors, ligands, cell adhesion molecules,enzymes, peptide hormones and immune system proteins.

[0109] The vasculogenic progenitor cells of the present invention may bemanipulated to express exogenous polypeptides by introduction of anucleotide sequence encoding the exogenous polypeptide, or a precursorform of the exogenous polypeptide. Exogenous foreign nucleic acidsequences can be transferred to the vasculogenic progenitor cells of theculture by electroporation, calcium phosphate, microinjection,lipofection, retro- or other viral or microbial vector or other meanswell known to one of ordinary skill in the art. Preferably, expressionof the exogenous sequence(s) is inducible. Cells expressing theexogenous polypeptide may be screened and isolated by techniques wellknown in the art including, but not limited to immunoblotting,immunofluorescence, ELISA and RT-PCR. Cells expressing exogenouspolypeptides can be harvested, expanded, differentiated and used for,for example, repairing or augmenting a defect. In this manner, cells,tissues or organs can be prepared with exogenous majorhistocompatability antigens which will decrease rejection oftransplanted materials by the host organism. In addition, cellsexpressing and secreting vasculogenic growth factors, or overexpressinggrowth factor receptors can be selected and cultured, creating culturesof vasculogenic progenitor cells with altered temporal dynamics and/orsensitivities to differentiation factors.

[0110] The vasculogenic progenitor cells isolated by the methods of thepresent invention can be used therapeutically, in treatment of vascularand vascular related disease. Potential applications include celltransplantation for repair of damaged and ischemic tissues,vascularization of regenerating tissue and embryonic regenerativemedicine. Examples of such therapeutic applications of stem andprogenitor cells are the augmentation of vessel growth observed in areasof ischemic tissue after implantation of adult endothelial progenitors(Kawamoto A et al Circulation 2001;103:634-37) and theneovascularization by adult endothelial progenitors following cerebralischemia in induced stroke in mice (Zhang Z G et al Circ Res2002;90:284-88).

[0111] Thus, according to yet another aspect of the present inventionthere is provided a method of relieving or preventing a vascular diseaseor condition in a mammalian subject. The method is effected byadministering the vasculogenic progenitor cells of the present inventionto the subject. Methods of administering the progenitor cells of thepresent invention to subjects, particularly human subjects includeinjection or implantation of the cells into target sites in thesubjects, the cells of the invention can be inserted into a deliverydevice which facilitates introduction by, injection or implantation, ofthe cells into the subjects. Such delivery devices include tubes, e.g.,catheters, for injecting cells and fluids into the body of a recipientsubject. In a preferred embodiment, the tubes additionally have aneedle, e.g., a syringe, through which the cells of the invention can beintroduced into the subject at a desired location. The progenitor cellsof the invention can be inserted into such a delivery device, e.g., asyringe, in different forms. For example, the cells can be suspended ina solution or embedded in a support matrix when contained in such adelivery device. As used herein, the term “solution” includes a carrieror diluent in which the cells of the invention remain viable. Carriersand diluents which can be used with this aspect of the present inventioninclude saline, aqueous buffer solutions, solvents and/or dispersionmedia. The use of such carriers and diluents is well known in the art.The solution is preferably sterile and fluid to the extent that easysyringability exists. Preferably, the solution is stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention canbe prepared by incorporating progenitor cells as described herein in acarrier or diluent and, as required, other ingredients enumerated above,followed by filtered sterilization.

[0112] Support matrices in which the vasculogenic progenitor cells canbe incorporated or embedded include matrices which arerecipient-compatible and which degrade into products which are notharmful to the recipient. Natural and/or synthetic biodegradablematrices are examples of such matrices. Natural biodegradable matricesinclude plasma clots, e.g., derived from a mammal, polymeric scaffolds,matrigel and collagen matrices. Synthetic biodegradable matrices(scaffolds) include synthetic polymers such as polyanhydrides,polyorthoesters, and polylactic acid. Other examples of syntheticpolymers and methods of incorporating or embedding cells into thesematrices are known in the art. See e.g., U.S. Pat. Nos. 4,298,002 and5,308,701. These matrices provide support and protection for the fragileprogenitor cells in vivo and are, therefore, the preferred form in whichthe vasculogenic progenitor cells are introduced into the recipientsubjects.

[0113] Differentiation of the implanted vasculogenic progenitor cells ofthe invention may be directed by factors originating from thesurrounding tissue, or may be initiated by pre-implantation incubationwith lineage-specific growth factors. Thus, for example, defectsrequiring regeneration of smooth muscle can be treated with cells havingbeen exposed to PDGF-BB, to achieve a population enriched in smoothmuscle precursors.

[0114] Vascular disease and conditions that can be treated with themethods of the present invention include congenital and acquiredvascular disorders and ischemia/reperfusion injury. As used herein, theterm “congenital vascular disorders” refers to vascular disordersexisting from birth, including both hereditary and developmentaldisorders. “Acquired vascular disorders” refers to vascular disordersensuing after birth, including secondary vascular manifestations ofsystemic or other disease, such as the microvascular pathologies ofdiabetes. “Ischemia/reperfusion injury” refers to cell or tissue injuryresulting from interrupted or diminished blood supply, and the tissuedamage, especially the inflammatory response, associated withreestablishing circulation in ischemic tissues.

[0115] Conditions which may benefit from such treatment include ischemicconditions (associated, for example, with myocardial, brain orperipheral vascular ischemia), wound healing, tissue grafting (includingtransplant) and conditions involving endothelial cell growth andproliferation, for example after coronary angioplasty, stenting orrelated procedures, re-endothelialization of arterial grafts, andendothelial regeneration in A-V shunts, e.g. in renal dialysis patients.In view of the complications encountered using porcine progenitorxenografts in primates (Buhler L et al Transplantation 2000; 70:1232-31)the methods of the present invention, which can be applied to human stemcells, are especially suited for treatment of such vascular conditions.

[0116] Since it was observed herein that the vasculogenic progenitorcells of the invention, when exposed to hematopoietic growth factors andcytokines, can be induced to differentiate into blood cell progenitorsand mature blood cells (FIGS. 2K-M and 3D-F, respectively), thevasculogenic progenitor cells of the invention can also be used fortreating or preventing a hematological disease or condition in amammalian subject.

[0117] Such treatment can be effected by administering the cells into asubject under conditions suitable for stimulating differentiation intoboth endothelial and blood cells. Hematological diseases or conditionsthat can be treated or prevented in this manner include congenital andacquired blood disorders, clotting disorders and neoplastic disease.

[0118] One example of a clotting disorder suitable for treatment by themethod of the present invention is von Willebrand's disease, a type ofhemophilia caused by deficiency in the endothelial von Willebrandclotting factor. While reducing the present invention to practice, itwas uncovered that differentiated endothelial cells prepared by themethods of the present invention contain von Willebrand factor (FIGS.3A-G and 4A-B). Thus, endothelial cells or endothelial progenitor cellsof the present invention, or compositions thereof can be administered,producing the clotting factor and alleviating the clotting deficiency.

[0119] In addition to the abovementioned therapeutic applications,vasculogenic progenitor cells isolated and prepared by the methods ofthe present invention can be used to provide vascularization ofnon-vascular, or inherently poorly vascularized tissue. It will beappreciated that one of the most important challenges facing the fieldof tissue engineering is the adequate perfusion of tissue and organsprepared in vitro for implantation. To date, most tissue engineeringmethods have relied on microporous supports and vascularization from thehost to provide permanent engraftment and transfer of oxygen andnutrients, with varying and often unpredictable results, especiallywhere thick, complex tissues (e.g. liver) are concerned. One alternativeapproach is the fabrication of “vascular” channels in silicon bymicromachining, for population by mixed hepatocytes and endothelialcells in vitro (Kaihara S et al Tissue Eng 2000;6:105-07). In anotherapproach more closely mimicking normal development, endothelial cellshave been cocultured with skin (Black A F et al Cell Biol Toxicol1999;15:81-90) or adipose (Frerich B et al Int J Oral Maxillofac Surg2001;30:414-20) cells to provide a vascular network for the growingtissue. However, none have been successful in engineering viable,implantable vascularized tissues.

[0120] Thus, according to a further aspect of the present invention,there is provided a method of vascularizing a mammalian tissue. Themethod is effected by obtaining a population of vasculogenic progenitorcells and contacting the cells with a mammalian tissue under conditionssuitable for differentiation of the vasculogenic progenitor cells intoendothelial and smooth muscle cells. In one preferred embodiment, themammalian tissue is an engineered, non-vascular tissue.

[0121] Examples of such engineered tissue are masses of in vitroprepared hepatocytes, epidermal and dermal cells, pancreatic and bonecells for implantation. Contacting the tissue with the differentiatingvasculogenic progenitor cells can be by coculture in semisolid matrix oron a porous scaffold, as is commonly used in engineered tissuearchitecture, as detailed hereinabove. Contacting the mammalian tissuecan be performed in vitro, prior to implantation into the host organism,or in vivo, into a previously implanted or existing tissue. In anotherpreferred embodiment, the mammalian tissue is an embryonic tissue,prepared for implantation into adult host organism, or for implantationand growth as an embryo.

[0122] While reducing the present invention to practice, it was alsoobserved that the cells larger than 50 μm retained by the size selectionstep of the present methodology comprise a population enriched in smoothmuscle cells precursors, expressing characteristic epithelial cellmarkers and morphology (FIGS. 1H and 1G, respectively).

[0123] Thus, the present invention also provides a method of preparingepithelial progenitor cells from undifferentiated ES cells. The methodis effected by culturing the undifferentiated ES cells in a mannersuitable for differentiation into vasculogenic progenitor cells andisolating cells larger than 50 μm. Conditions for culture of theepithelial precursors, and their differentiation into smooth musclecells, were substantially similar to those detailed herein for thevasculogenic progenitor cells, with substitution of smooth muscle orepithelial growth factors, such as PDGF-BB, in place of endothelial orvasculogenic growth factors. However, it was noted that the epithelialand smooth muscle cells lack the proliferative capacity of the smaller,vasculogenic progenitor cells.

[0124] Adult vascular tissue is comprised of endothelial and epithelialcells, distinguishable by size, morphology and cell markers, as well aslocation and function. Current methods for the isolation of vascularcell types rely upon cell surface marker detection, immunofluorescenceand flow cytometry (see, for example, Kevil E G and Bullard D C ActaPhysiol Scand 2001;173:151-57), making the preparation of vascular cellsfor experimentation and primary culture cumbersome, expensive andinefficient. Thus, it will be appreciated that the methods of thepresent invention may be employed to isolate and prepare cells fromvascular tissue, as well as from undifferentiated stem cells. In apreferred embodiment, the cells of the vascular tissue are dissociatedby mechanical, or enzymatic means, such as trypsin or collagenasedigestion, to obtain a mixed population of dissociated cells, and thesmaller (smaller than 50 μm) endothelial cells isolated by sizeselection as detailed herein for the vasculogenic progenitor cells.Similarly, adult epithelial cells can be isolated from vascular tissueby a similar method, wherein the retained cell population (greater than50 μm), rich in epithelial cells, is collected.

EXAMPLES

[0125] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

[0126] Generally, the nomenclature used herein and the laboratoryprocedures utilized in the present invention include molecular,biochemical, microbiological and recombinant DNA techniques. Suchtechniques are thoroughly explained in the literature. See, for example,“Molecular Cloning: A laboratory Manual” Sambrook et al., (1989);“Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M.,ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”,John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guideto Molecular Cloning”, John Wiley & Sons, New York (1988); Watson etal., “Recombinant DNA”, Scientific American Books, New York; Birren etal. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

[0127] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below.

[0128] Materials and Methods

[0129] Cell Culture

[0130] Undifferentiating human embryonic stem (hES) cells (H9.2, H13)were grown on inactivated mouse embryonic feeder layer (MEF) aspreviously described (Amit M, et al. Dev Biol 2000; 227: 271-78), in 80%knock-out DMEM medium (no pyruvate, high glucose formulation; LifeTechnologies Inc., Rockville, Md. USA) supplemented with 20% FBS(HyClone, Logan, Utah, USA), or serum replacement and bFGF, 1 mML-glutamine, 0.1 mM mercaptoethanol, and 1% nonessential amino acidstock (Life Technologies Inc., Rockville, Md., USA). hES cells wereremoved from the feeder layer using EDTA 5 mM supplemented with 1% fetalbovine serum (FBS; HyClone, Logan, Utah, USA) and dispersed tosingle-cells using a 40 μm mesh strainer (Benton, Dickinson and Co,Discovery Labware, Bedford, Mass., USA).

[0131] For differentiation, undifferentiated hES single cells wereplated on type IV collagen-coated (Becton Dickinson and Co, San Jose,Calif., USA) or 0.1% gelatin-coated (Sigma Chemical Co., St Louis Mo.,USA) 6-well dishes at a concentration of 5×10⁴ cells/cm², indifferentiation medium consisting of alpha MEM medium (Life TechnologiesInc., Rockville, Md., USA) supplemented with 10% FBS (HyClone, Logan,Utah, USA) and 0.1 mM β-mercapoethanol (Life Technologies Inc.,Rockville, Md., USA). On day 6 of culture cells were filtered through a40 μm mesh strainer (Becton, Dickinson and Co, Discovery Labware,Bedford, Mass., USA) and were analyzed or recultured for furtherdifferentiation. For reculture, the strained cells were seeded at2.5×10⁴ cells/cm² on type IV collagen coated dishes (Benton Dickinsonand Co, San Jose, Calif., USA) in differentiation medium (see above)with hVEGF₁₆₅ 50 ng/ml or hPDGF-BB 10 ng/ml (both from R&D Systems Inc,Minneapolis, Minn., USA) for an additional 10-12 days.

[0132] Collagen gel and Matrigel 3-D vascularization assays Before threedimensional culture, filtrated cells cultured for 6 days indifferentiation medium were harvested with EDTA 5 mM and 0.3-0.5×10⁶cells per ml were incubated in differentiation medium containing 50ng/ml VEGF₁₆₅ and hPDGF-BB 10 ng/ml on uncoated petri dishes (Ein-ShemerIndustries, Israel) for maximum of 24 hours to induce aggregation. Forthe collagen gel assay, aggregates were resuspended in 2×differentiation medium and mixed with an isovolume of rattail collagen 1(3 mg/ml) (F.Hoffman-La Roche Ltd, Basel, Switzerland). Initially, 250μl of this mixture was plated in 24-well dishes, which was allowed topolymerize for 15 min at 37° C., before adding 500 μl of differentiationmedium supplemented with the same growth factors. For the Matrigelassay, 24-well dishes were coated with 380 μl of Matrigel (BectonDickinson and Co, San Jose, Calif., USA), incubated 30 min at 37° C.,and aggregates were seeded on the matrigel in differentiation mediumcontaining hVEGF (50 ng/ml) and hPDGF-BB (10 ng/ml). In some assays,aggregates were resuspended within the Matrigel (Becton Dickinson andCo, San Jose, Calif., USA), incubated for 30 min at 37° C., and thenadded to the wells with differentiation medium containing hVEGF (50ng/ml) and hPDGF-BB (10 ng/ml). For all assays, cells were incubated for7-12 days and analyzed using contrast-phase microscope (Olympus OpticalCo Ltd, Hamburg GmbH).

[0133] Scaffold Vascularization

[0134] LF120 50 μl alginate scaffold (Shapiro L and Cohen S.Biomaterials 1997;18: 583) was kindly provided by Prof Smadar Cohen (BenGurion University, Beer Sheba, Israel). As described above, thescaffolds were seeded with 24 hour old ESH cell aggregates prepared asdescribed hereinabove; approximately 0.5-1.0×10⁶ cells were seeded perscaffold. The cell-containing scaffolds were then cultured indifferentiation medium supplemented with 50 ng/ml VEGF₁₆₅ and 10 ng/mlhPDGF-BB.

[0135] Hematopoietic Colony Assay:

[0136] Hematopoietic progenitor capability was demonstrated by seedingfiltrated, VEGF-treated, VE-cad+ ESH cells, as single cells, 1-2×10⁵cells per plate, in semisolid media supplemented with cytokines(Methocult GF+ media; StemCell Technologies, Vancouver BC) (for detailsof the assay, see Kaufman D S et al, PNAS 2001;98:10716-21). After 14days incubation, the plates were scored for colony-forming units (CFU),according to standard criteria [Eaves C and Lambie, K Atlas of HumanHematopoietic Colonies (1995); StemCell Technologies, Vancouver, BC].

[0137] Immunostaining, Dil-Ac-LDL and BrdU incorporation Cultured cellswere fixed in situ by incubation with 4% paraformaldehyde (Sigma-AldrichCorp., St Louis, Mo., USA) in phosphate buffered saline (PBS) (LifeTechnologies Inc., Rockville, Md., USA) for 30 min at room temperature.After washing with PBS, cells were stained according to suppliersinstructions with relevant primary antibodies: goat anti human KDR (R&DSystems Inc, Minneapolis, Minn., USA), mouse anti hCD31, mouse antihSMA, mouse anti hCalponin, mouse anti h Smooth muscle myosin heavychain (all from DAKO Corp, Carpenteria, Calif., USA), goat anti humanVE-Cadherin (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA),(DAKO Corp, Carpenteria, Calif., USA), mouse anti Smoothelin (CHEMICON,Intn'l, Inc. Temecula, Calif., USA). Controls consisted of cellsincubated with secondary antibodies alone. Immunostained cultures wereexamined and photographed using fluorescence microscopy (Olympus OpticalCo, Ltd. Hamburg, GmbH).

[0138] For uptake of Dill-labeled ac-LDL, cultured ESH cells wereincubated with 10 μg/ml Dill-labeled ac-LDL (Biomedical TechnologiesInc., Stoughton, Mass., USA) for 4 h at 37° C. Following incubation,cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for30 minutes, examined and photographed using a fluorescent microscope(Olympus Optical Co, Ltd. Hamburg, GmbH).

[0139] BrdU incorporation in ESH cultures and differentiating cells wasexamined using a BrdU staining kit (Zymed Labs Inc., South SanFrancisco, Calif., USA) in-situ, according to manufacturersinstructions. Briefly, BrdU solution was diluted 1:100 in culture mediumand added to the cells overnight, followed by two PBS washes, fixationwith 75% ethanol and specific BrdU immunostaining.

[0140] Immunophenotype

[0141] Cells were characterized using immunofluorescence staining aspreviously described (Reubinoff B E et al., Nat Biotech 2001;19: 1134).Briefly, filter-separated ESH cells were recultured, as described above,in differentiation medium (alpha MEM, 10% FBS and 0.1 β-mercaptoethanol)on type IV collagen plates for 12-20 hours, fixed and assayed forexpression of specific cell-type markers with anti human VE-Cadherin(Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA), and antihuman KDR (R&D Systems Inc, Minneapolis, Minn., USA). At least onehundred and fifty cells were scored within random fields (×100) for theexpression of each of these markers, and experiments were repeated atleast three times.

[0142] Histolomorphology and Immunohistochemical Analysis

[0143] Matrigel or collagen gel containing cells (as describe for theCollagen gel and Matrigel 3-D vasculanization assays hereinabove) wereplated as described above onto glass cover slips, in 24-well dishes.Upon completion of treatments, the cell-containing gel blocks on coverslips were fixed in 10% neutral-buffered formalin, dehydrated ingraduated alcohol baths (70%-100%), and embedded in paraffin. Whereused, the alginate scaffolds were directly dehydrated in graduatedalcohol. For general histomorphology, 1-8 μm sections were stained withhematoxylin/eosin or toluidine blue. Deparaffinized sections wereimmunostained with the relevant primary antibodies, using LSAB+ stainingkit (DAKO Corp, Carpenteria, Calif., USA) or Cell and Tissue stainingkit (R&D Systems Inc, Minneapolis, Minn., USA) according tomanufacturers instructions. Stained sections were viewed andphotographed microscopically at ×100-×400 magnification.

[0144] FACS Analysis

[0145] Cells expressing the endothelial progenitor markers VEGFR2 (KDR)and VE-cad were detected and quantified from the two size-separatedhuman ES cell populations after filtration and separate reculturing ontype IV collagen, as described above. For FACS analysis, ESH filteredcells were washed in PBS containing 5% FBS, incubated with humanVE-Cadherin (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA),or human KDR (R&D Systems Inc, Minneapolis, Minn., USA), washed, andincubated 30 min with suitable second antibodies. Cells were analyzedusing a FACSCalibur (Benton Dickinson and Co, San Jose, Calif., USA)with CELLQUEST software. IN both assays, cells reacted with secondantibodies only served as controls.

[0146] Electron Microscopy

[0147] Cell seeded in Matrigel or collagen gel were fixed for one hourin 3% glutaraldehyde, in 0.1M sodium cacodylate and then post-fixed in1% OsO₄ in veronal-acetate buffer for 1 hour. Preparation for electronmicroscopy analysis was performed according to standard procedure at thePathology Department of the Rambam Medical Center, Haifa, Israel.Briefly, the cells were stained with lead-citrate, dehydrated andembedded in Epon resin. Sections were cut at a thickness of 600 Å usinga diamond knife, examined and photographed using a JEM-100SX electronmicroscope.

[0148] Reverse Transcription (RT)-PCR Analysis

[0149] Total RNA was extracted from progenitors and different lineagecells using TriReagent (Sigma-Aldrich Corp., St Louis, Mo., USA)according to the manufacturer's instructions. Total RNA was quantifiedby UV spectrophotometry, and 1 μg was used for each RT sample. RNA wasreverse transcribed with M-MLV Reverse Transcriptase (Promega Corp.,Madison, Wis., USA) and oligo (dT) primers (Promega Corp., Madison,Wis., USA) according to manufacturer's instructions. PCR amplificationof the selected transcripts was done with BIOTAQ™ DNA Polymerase(BIOLINE, Ltd GmbH Luckenwalde, Germany) using 1 μl of RT product perreaction, according to manufacturers instructions. In some cases MgCl₂concentration (normally 1.5 mM) was calibrated (indicated below). Toensure semi-quantitative results in the RT-PCR assays, the number of PCRcycles for each set of primers was verified to be in the linear range ofthe amplification. In addition, all RNA samples were adjusted to yieldequal amplification of the housekeeping gene GAPDH as an internalstandard. PCR conditions and protocol consisted of: 5 min at 94° C. (hotstart); followed by 30-40 cycles (actual number noted below) of: (a) 94°C. for 30 sec; (b) annealing temperature (Ta, noted below) for 30 sec;and (c) 72° C. for 30 sec, concluding with a final 7-min extension at72° C. at the end. Oligonucleotide-specific conditions were as follows:a-sma, 32cycles, Ta 60° C. (Yamamura, H et al. Int. J. Cancer (Pred.Oncol.) 1998;79: 245); calponin, 35cycles, Ta 60° C. (Yamamura, H et al.Int. J. Cancer (Pred Oncol) 1998; 79: 245); SM-MHC, 35cycles, Ta 62° C.,1 mM MgCl₂ (Boreham et al. Am J Obsetet Gynycol 2001;185:944-52); SM22α,35cycles, Ta 60° C. (Yamamura, H et al. Int. J. Cancer (Pred. Oncol.)1998;79: 245); caldesmon, 35cycles, Ta 60° C. (Duplaa, C. et al., CircRes. 1997;80:159); GATA2, 35cycles, Ta 55° C. (Kaufman D S et at PNAS2001; 98:10716); AC133, 32cycles, Ta 60° C. (Shamblott M J et at, PNAS2001;98:113); Tie2, 35cycles, Ta 60° C. (Ahmad,S et al, Cancer2001;92:1138); CD31, 32cycles Ta 60° C. (Quarmby, S et al ArterioThrombo Vas Biol 1999;19:588-97); Tall, 40 cycles Ta 53° C. (Kaufman D Set al PNAS 2001; 98:10716); GAPDH, 32cycles, Ta 60° C. (Itskovitz-EldorJ et at Mot Med 2000;6:88).

[0150] Oligonucleotide Primers:

[0151] For the PCR reactions the following specific oligonucleotideprimers were used:

[0152] (a) α-sma: 5′ CCAGCTATGTGAAGAAGAAGAGG 3′ (SEQ. ID. NO: 1) (sense)and 5′ GTGATCTCCTTCTGCATTCGGT 3′ (SEQ. ID. NO: 2) (antisense). Thepredicted size of band is 965 base pairs;

[0153] (b) Calponin: 5′ GAGTGTGCAGACGGAACTTCAGCC 3′ (SEQ. ID. NO: 3)(sense) and 5′ GTCTGTGCCCAACTTGGGGTC 3′ (SEQ. ID. NO: 4) (antisense).The predicted size of band is 671 base pairs;

[0154] (c) SM-MHC: 5′ CTACAGGAGCATGCTGCAGGATCG 3′ (SEQ. ID. NO: 5) and5′ GCTTGCAGAAGCTGCTTCTCCAGC 3′ (SEQ. ID. NO: 6), corresponding tonucleotides 579 (sense) and 758 (antisense), respectively. The predictedsize of band is 179 base pairs;

[0155] (d) SM22α: 5′ CGCGAAGTGCAGTCCAAAATCG 3′ (SEQ. ID. NO: 7) (sense)and 5′ GGGCTGGTTCTTCTTCAATGGGG 3′ (SEQ. ID. NO: 8) (antisense). Thepredicted size of band is 928 base pairs;

[0156] (e) Caldesmon: 5′ AACAACCTGAAAGCCAGGAGG 3′ (SEQ. ID. NO: 9) and5′ GCTGCTTGTTACGTTTCTGC 3′ (SEQ. ID. NO: 10), corresponding tonucleotides 244 (sense) and 792 (antisense), respectively. The predictedsize of band is 530 base pairs;

[0157] (f) GATA2: 5′ AGCCGGCACCTGTTGTGCAA 3′ (SEQ. ID. NO: 11) (sense)and 5′ TGACTTCTCCTGCATGCACT 3′ (SEQ. ID. NO: 12) (antisense). Thepredicted size of band is 242 base pairs;

[0158] (g) AC133: 5′ CAGTCTGACCAGCGTGAAAA 3′ (SEQ. ID. NO: 13) (sense)and 5′ GGCCATCCAAATCTGTCCTA 3′ (SEQ. ID. NO: 14) (antisense). Thepredicted size of band is 200 base pairs;

[0159] (h) Tie2: 5′ ATCCCATTTGCAAAGCTTCTGGCTGGC 3′ (SEQ. ID. NO: 15)(sense) and 5′ TGTGAAGCGTCTCACAGGTCCAGGATG 3′ (SEQ. ID. NO: 16)(antisense). The predicted size of band is 512 base pairs;

[0160] (i) CD31: 5′ CAACGAGAAAATGTCAGA 3′ (SEQ. ID. NO: 17) (sense) and5′ GGAGCCTTCCGTTCTAGAGT 3′ (SEQ. ID. NO: 18) (antisense). The predictedsize of band is 260 base pairs;

[0161] (j) Tall: 5′ ATGGTGCAGCTGAGTCCTCC 3′ (SEQ. ID. NO: 19) (sense)and 5′ TCTCATTCTTGCTGAGCTTC 3′ (SEQ. ID. NO: 20) (antisense). Thepredicted size of band is 331 base pairs;

[0162] (k) GAPDH: 5′ AGCCACATCGCTCAGACACC 3′ (SEQ. ID. NO: 21) (sense)and 5′ GTACTCAGCGGCCAGCATCG 3′ (SEQ. ID. NO: 22) (antisense). Thepredicted size of band is 302 base pairs.

EXAMPLE 1 Isolation and Enrichment of Human Vasculogenic ProgenitorCells from Human Stem Cells

[0163] Despite the overwhelming importance of human stem cell technologyto research and medicine, application of discoveries made in researchwith non-human species to human stem cells has been painstakinglydifficult, requiring great ingenuity and much effort. While murineembryonic stem cell (mES) lines, for example, retain their pluripotencyin culture, and may be predictably manipulated to differentiate in vitrointo cells of mesodermal, endodermal and ectodermal lineage, in vitrodifferentiation in human and other primate ES cell lines has beencharacterized by inconsistency, disorganization, and lack of synchrony,obviating successful in vitro tissue organization (see, for example,Thompson, et al Curr Top Dev Biol 1998; 38:133-165). In pursuing theisolation of vasculogenic progenitor cell from human embryonic stemcells, initial human ES mesodermal differentiation was attempted in anovel two dimensional (2D) rather than the three dimensional (3D) modelcommonly used in the art, based upon the observation that the 3Dembryoid body structure is not required for mouse stem cell mesodermaldifferentiation (Nishikawa S-I., et al., Development 1998; 125:1747).

[0164] Undifferentiated human embryonic stem cell line H9.2 and H13cells were grown as previously described (Amit M et al Dev Biol2000;227:271-78), removed from feeder layer and plated as single cellson type IV collagen coated dishes with differentiation medium as hadbeen described for mouse CCE-ES cells (Yamashita J, et al. Nature 2000;408:92). Previous experience with murine stem cells indicated thatspecific cell seeding concentration is crucial for induction endothelialdifferentiation. However, seeding human ES cells in the recommended cellconcentration (1×10⁴ cells/cm²) resulted in cell death. Therefore,several cell-seeding concentrations were investigated. Seeding the cellsat higher concentrations on a variety of attachment substrates(1.0-1.5×10⁵ cells/cm²) resulted in an inconsistent mixed population ofundifferentiated and differentiating cells (data not showed).Surprisingly, seeding cells at low concentration (5-10×10⁴ cells/cm²) ontype IV collagen substrate promoted differentiation that resulted in twodistinct populations of cell types (FIG. 1B). A significant proportionof the cell population comprised smaller flat cells with large nucleiresembling endothelial progenitor morphology (FIG. 1B, arrows)previously recognized in murine cells (Yamashita J, et al. Nature 2000;408:92), while the remainder were large flat cells with obvious fibrousstructure (FIG. 1B, arrowheads).

[0165] In order to separate the two cell populations, and isolate thesmaller, human vasculogenic cell progenitors, the cells were filteredthrough a 40 μm strainer, segregating the endothelial-like cells fromthe large flat cells. To evaluate the proportion of endothelialprogenitors in the cultures, the filtered cell populations werecharacterized by detection of specific cell-type markers, as previouslydescribed for monitoring the differentiation of neuron progenitorsderived from hES cells (Reubinoff BB et al., Nat Biotech 2001;19:1134).Filtrated cells were plated, fixed, and analyzed immunologically for theexpression of human vascular endothelial endothelial receptor 2 (VEGFR2,KDR), and vascular endothelial cadherin VE-cad, both known to play animportant role in mouse endothelial progenitor development (NishikawaS-I., et al., Development 1998; 125: 1747).

[0166] Unexpectedly, when the expression of these markers in the twopopulations was quantified by immunodetection and FACS analysis (FIGS.1C-E), a significant proportion of the smaller, endothelial-like cellswere found to express VE-cad (78%, FIG. 1C) and a smaller portionexpressed VEGFR2 (28%, FIG. 1C). When the smaller, filtrated cells wereplated for 12 hours, fixed and analyzed for immunomorphology withfluorescent anti VE-cad antibody (FIGS. 1D-E), significantly greaterexpression of VE-cad was detected (90.55+5.20%), most likely due to thelow fluorescent intensity observed when VE-cad is expressed at thecell-to-cell junctions (FIG. 1E). Trials of a variety of cell-seedingdensities indicated optimal VE-cad expression at 5×10⁴ cells/cm².

[0167] When further characterized by RT-PCR amplification of RNA, theendothelial-like cells were found to actively express the endothelialmarkers CD31 and Tie2; AC133/CD133, GATA2 and Tall, earlyendothelial/hematopoeitic progenitor cell markers (Peichev, M et al.,Blood 2000; 95:952 and Kaufman D S et al PNAS 2001; 98:10716) (FIG. 1F,Filtrated). RT-PCR of RNA from undifferentiated human stem cells (FIG.1F, hES) demonstrated no CD31, Tie2, Tall or GATA2 expression, and onlyminimal expression of AC133. Note that the intensities of the GAPDHbands are identical for both the undifferentiated and differentiatedcell populations (FIG. 1F), indicating the specific nature of the changein cell phenotype with differentiation.

[0168] Immunofluorescent staining of the larger, excluded cells (FIG.1G) revealed the existence of epithelioid phenotype smooth muscle cellfeatures (reviewed by Gittenberger-de Groot A. C, et al PNAS2000;97:11307) and markers (αSMA) undetected in the smaller, filteredcells. When further characterized by RT-PCR amplification of RNA, thelarger, excluded cells were found to actively express epitheliod markersCalponin and Caldesmon; smooth muscle actin (SMA), and SM-MHC (FIG. 1H,Retained). RT-PCR of RNA from the smaller, endothelial-like cellsdemonstrated no expression of any of the epitheliod cell markers (FIG.1H, Filtrated). Note that the intensities of the GAPDH bands areidentical for both the cell populations (FIG. 1H), indicating thespecific nature of the change in cell phenotype with differentiation.

[0169] When the two cell populations arising from the low-densityseeding, and culturing of human stem cells (hES) were assessed for cellproliferation capability, the BrdU incorporation assay revealed that theepitheliod, excluded large smooth muscle-like cells are unable toproliferate (FIG. 1I, arrow) while the smaller, endothelial-likeprogenitor cells clearly incorporate the stain, indicating retention ofproliferative ability (FIG. 1I). Taken together, these results indicatethat human stem cells, seeded as single cells and not as EmbryoidBodies, and cultured in vitro on a cell-free, two-dimensional matrix,can give rise to proliferating, endothelial-like progenitor cells, whichcan be separated by filtration from smooth muscle-like precursors.

EXAMPLE 2 In Vitro Induction of Endothelial, Smooth Muscle andHematopoietic Cell Differentiation of Human Vasculogenic ProgenitorCells

[0170] In order to study the differentiation potential of thevasculogenic progenitor cells, cells were recultured on type IV collagencoated dishes, at a lower cell seeding concentration (2.5×10⁴cells/cm²). Smooth muscle cell differentiation was induced by addingplatelet-derived growth factor BB (hPDGF-BB), which has been found toinduce SMC differentiation in murine (mES), but not human stem cells(Gittenberger-de Groot A. C et al PNAS 2000;97:11307). After 10-12 daysof culture both spindle-like shaped and epithelioid phenotype cells weredetected in the culture, along with a concomitant induction ofexpression smooth muscle cell markers. RT-PCR analysis detectedupregulation of specific smooth muscle markers such as smooth muscleα-actin (SMA), smooth muscle myosin heavy chain (SM-MHC), calponin,SM22, and caldesmon (FIG. 2A, v-SMC), notably undetectable in the RNAfrom non-hPDGF-BB treated cells (FIG. 2A, ESH progenitor cells).Immunofluorescent detection of the human smooth muscle cell markerproteins (ASMA, FIG. 2B; smoothelin, a marker of early smooth muscledevelopment, FIG. 2C; SM-MHC FIG. 2D and Calponin FIG. 2E) confirms thecapacity for further in vitro differentiation of human vasculogenicprogenitor cells by exposure to hPDGF-BB.

[0171] To test the potential of differentiation to endothelial cells,the human vasculogenic progenitor cells were exposed to hVEGF₁₆₅, foundto be efficient in murine, but not human endothelial cell induction(Yamashita J, et al. Nature 2000; 408:92). This manipulation resulted inthe induction of endothelial cell-specific markers: continuousexpression of VE-cad and the appearance of von Willebrand Factor (vWF)stored in Weibel-Palade bodies, as detected by immunofluorescence (FIGS.2F and 2G, respectively), Dill-Ac-LDL uptake in more mature cells (FIG.2H) and even stress fibers arrangement in some mature cells (FIG. 2I).Most significantly, growth factor-induced differentiation, with eitherhPDGF-BB or hVEGF₁₆₅, did not induce a lineage-specific commitment,i.e.: both endothelial and smooth muscle cell types were observed withadministration of each of the growth factors. Furthermore, BrdUincorporation into the differentiated cells indicated preservation ofproliferative capability in the vascular endothelium growth-factor(VEGF) treated cells (FIG. 2I), and specifically those cells of thesmaller morphology, while cells treated with hPDGF-BB exhibited impairedproliferation ability (FIG. 2J). Hematopoietic capability of theisolated progenitor cells was also demonstrated. When theVE-cad-expressing population of filtrated, vasculogenic progenitor cellswas cultured in a semisolid medium with cytokines, CFUs indicatinghematopoietic colonies (FIGS. 2K-M) were observed. Thus, differentiationof isolated human vasculogenic progenitor cells may be further induced,and controlled, by specific growth factors in vitro, in a cell-freemedium, without lineage-specific commitment or loss of proliferativecapability.

EXAMPLE 3 In-vitro Vasculogenesis and Blood Cell Formation by ESH Cells

[0172] Crucial events characteristic of vasculogenesis have been inducedin vitro using murine embryonic stem cell-derived embryoid bodies (see,for example, Feraud O et al Lab Investig 2001;81: 1661-89), howeverefforts to emulate vasculogenic processes in vitro using humanpluripotent stem cells have been largely unsuccessful. To study thein-vitro vascularization potential of human vasculogenic progenitor(ESH) cells we used two different 3-dimensional models: type I collagengel and Matrigel, which have been used to promote 3D vessel-likeformation from endothelial cells (Mardi JA and Pratt B M, B.M.J CellBiol.1988; 106:1375; Kubota Y et al J Cell Biol 1988;107:1589).

[0173] Aggregation of the ESH cells, in the presence of hVEGF andhPDGF-BB supplemented differentiation medium, prior to seeding into typeI collagen (FIG. 3A) or on Matrigel (FIG. 3B) clearly induces sproutingand tube-like structures associated with early vasculogenesis andvascularization of both the collagen and Matrigel substrate.Histological sections demonstrate penetration of the endothelial cellsinto the Matrigel, forming a tube-like network structure characteristicof vascular formation (FIG. 3C). Surprisingly, and of great importance,observation under higher magnification reveals blood cells within thesein-vitro cultivated vessels (FIG. 3D, arrow). Electron microscopyfurther reveals well-formed endothelial-specific Weibel-Palade bodies(WP) in the cell cytoplasm, lipoprotein capsules (Li), endothelial cellsforming a lumen (Lu) in the cords and hematopoietic (BC) developmentwithin the vessels formed by endothelial cells (EC) within the Matrigel(M) (FIGS. 3E-3G). These results demonstrate, for the first time,isolated human vasculogenic progenitor cells having the capacity todifferentiate into functional endothelial cells with lipoproteinmetabolism, factor VIII (vWF) production, blood cells, and allcomponents of vascular structures in vitro, under defined conditions.

EXAMPLE 4 3 Dimensional Scaffold Vascularization

[0174] In vitro vascularization of engineered tissues is a criticalaspect of regenerative medicine, crucial for the maintenance of culturedtissue viability before and after implantation. Large-diameter vascularstructures, suitable for implantation, require a supporting framework,e.g. scaffold, for efficient development and growth. Therefore, thetherapeutic potential of human vasculogenic (ESH) progenitor cells wasinvestigated using an in-vitro tissue engineering model, the3-dimensional alginate scaffold, which has been shown to support invitro tissue formation from fibroblasts and hepatocytes (Shapiro L, andCohen S. Biomaterials 1997;18: 583; and Glicklis R, et al BiotechnolBioeng 2000;67:344), but not human vasculogenic progenitors.

[0175] When human vasculogenic progenitors were aggregated, asdescribed, and seeded within porous alginate scaffolds, distinct vesselformation around the scaffold pores was observed after 14 daysincubation in differentiation medium supplemented with both hVEGF andhPDGF-BB (FIG. 4A, red-staining structures). Higher magnificationexamination of the vascular wall structure reveals flat, elongatedendothelial cells surrounded by smooth muscle cells, typical of vascularmorphology (FIG. 4B). Thus, culturing human vasculogenic progenitor(ESH) cells on 3-D scaffolds demonstrated, for the first time, thecapability for directed, in-vitro vasculogenesis with differentiatedhuman stem cells, faithful to normal angiogenic development.

EXAMPLE 5 Human Vasculogenic Progenitor Cell Differentiation as a Modelfor Angiogenesis

[0176] Recent studies have demonstrated that some murine embryonic stemcell (mES) systems are capable of reproducing key events and chronologyof the angiogenic process, providing a potentially useful tool withwhich to investigate mechanisms of angiogenesis (Feraud, 0 et al Lab.Invest. 2001;81: 1669). Of further significance was the observation thatmES cells derived from VE-cad deficient strains of mice (VE-cad −/−)failed to develop endothelial sprouts. However, only embryoid bodies(mEB), and not single cells, were capable of initiation of thevasculogenic events in vitro.

[0177] To investigate whether directed, in-vitro vascular developmentfrom isolated human vasculogenic progenitor cells accurately reflectsphysiological processes of angio- and vasculogenesis, the effect ofinhibitory antibodies was assessed using BV6, a hVE-cad-specificmonoclonal antibody found to inhibit in vitro tube-formation of humanendothelial cells (Corada M et al., Blood 2001;97: 1679).

[0178] Suprisingly, the anti-VE-cad monoclonal exhibited a stronginhibitory effect on in vitro vascularization by ESH cells. 7 days afterincubation of ESH cells seeded on Matrigel in differentiation mediumsupplemented with growth factors, the vessels and network structurestypical of early vasculogenesis are clearly discernible in the gel (FIG.5A). Addition of 50 μg/ml of the anti hVE-cad antibody BV6 to the mediumclearly disrupted vasculogenesis, inhibiting essential cell sproutingand the formation of tube and network structures (FIG. 5B). Thus,in-vitro, directed differentiation of isolated human vasculogenicprogenitor (ESH) cells exhibits sensitivity to known inhibitors of humanangiogenesis-vasculogenesis, and as such, provides a model for studyingand assessing vascular-related effectors and therapies.

[0179] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1 22 1 23 DNA Artificial sequence Single strand DNA oligonucleotide 1ccagctatgt gaagaagaag agg 23 2 22 DNA Artificial sequence Single strandDNA oligonucleotide 2 gtgatctcct tctgcattcg gt 22 3 24 DNA Artificialsequence Single strand DNA oligonucleotide 3 gagtgtgcag acggaacttc agcc24 4 21 DNA Artificial sequence Single strand DNA oligonucleotide 4gtctgtgccc aacttggggt c 21 5 24 DNA Artificial sequence Single strandDNA oligonucleotide 5 ctacaggagc atgctgcagg atcg 24 6 24 DNA Artificialsequence Single strand DNA oligonucleotide 6 gcttgcagaa gctgcttctc cagc24 7 22 DNA Artificial sequence Single strand DNA oligonucleotide 7cgcgaagtgc agtccaaaat cg 22 8 23 DNA Artificial sequence Single strandDNA oligonucleotide 8 gggctggttc ttcttcaatg ggg 23 9 21 DNA Artificialsequence Single strand DNA oligonucleotide 9 aacaacctga aagccaggag g 2110 20 DNA Artificial sequence Single strand DNA oligonucleotide 10gctgcttgtt acgtttctgc 20 11 20 DNA Artificial sequence Single strand DNAoligonucleotide 11 agccggcacc tgttgtgcaa 20 12 20 DNA Artificialsequence Single strand DNA oligonucleotide 12 tgacttctcc tgcatgcact 2013 20 DNA Artificial sequence Single strand DNA oligonucleotide 13cagtctgacc agcgtgaaaa 20 14 20 DNA Artificial sequence Single strand DNAoligonucleotide 14 ggccatccaa atctgtccta 20 15 27 DNA Artificialsequence Single strand DNA oligonucleotide 15 atcccatttg caaagcttctggctggc 27 16 27 DNA Artificial sequence Single strand DNAoligonucleotide 16 tgtgaagcgt ctcacaggtc caggatg 27 17 18 DNA Artificialsequence Single strand DNA oligonucleotide 17 caacgagaaa atgtcaga 18 1820 DNA Artificial sequence Single strand DNA oligonucleotide 18ggagccttcc gttctagagt 20 19 20 DNA Artificial sequence Single strand DNAoligonucleotide 19 atggtgcagc tgagtcctcc 20 20 20 DNA Artificialsequence Single strand DNA oligonucleotide 20 tctcattctt gctgagcttc 2021 20 DNA Artificial sequence Single strand DNA oligonucleotide 21agccacatcg ctcagacacc 20 22 20 DNA Artificial sequence Single strand DNAoligonucleotide 22 gtactcagcg gccagcatcg 20

What is claimed is:
 1. A method of preparing vasculogenic progenitorcells from undifferentiated ES cells, the method comprising: (a)culturing individual undifferentiated ES cells in a manner suitable forinducing differentiation of the undifferentiated ES cells intovasculogenic progenitor cells, thereby obtaining a mixed population ofcells; and (b) isolating cells smaller than 50 μm from said mixedpopulation of cells, said cells smaller than 50 μm being vasculogenicprogenitor cells.
 2. The method of claim 1, wherein step (a) is effectedby subjecting the undifferentiated ES cells to at least one conditionselected from a group consisting of avoiding aggregation of ES cells,growth on collagen, cell seeding concentration between 2×10⁴ and 1×10⁵cells/cm² and presence of differentiation medium.
 3. The method of claim1, wherein said cells smaller than 50 μm are isolated via filtration,morphometry and/or densitometry.
 4. The method of claim 3, wherein saidfiltration is effected via a filter having a pore size smaller than 50μm.
 5. The method of claim 1, wherein said undifferentiated ES cells arehuman ES cells.
 6. A method of preparing epithelial progenitor cellsfrom undifferentiated ES cells, the method comprising: (a) culturingindividual undifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells thereby obtaining a mixed population of cells; and (b)isolating cells larger than 50 μm from said mixed population of cells,said cells larger than 50 μm being epithelial progenitor cells.
 7. Themethod of claim 6, wherein step (a) is effected by subjecting theundifferentiated ES cells to at least one condition selected from agroup consisting of avoiding aggregation of ES cells, growth oncollagen, cell seeding concentration between 2×10⁴ and 1×10⁵ cells/cm²and presence of differentiation medium.
 8. The method of claim 6,wherein said cells larger than 50 μm are isolated by filtration,morphometry and/or densitometry.
 9. The method of claim 8, wherein saidisolation by filtration is effected via a filter having a pore sizesmaller than 50 μm.
 10. The method of claim 6, wherein saidundifferentiated ES cells are human ES cells.
 11. A method of preparingsomatic cells from a population of vasculogenic progenitor cells, themethod comprising: (a) obtaining a population of vasculogenic progenitorcells; and (b) culturing the population of vasculogenic progenitor cellsin the presence of at least one growth factor suitable for inducingsomatic cell differentiation, thereby preparing the somatic cells. 12.The method of claim 11, wherein step (a) is effected by: (a) culturingindividual undifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells thereby obtaining a mixed population of cells; and (b)isolating cells smaller than 50 μm from said mixed population of cells,said cells smaller than 50 μm being vasculogenic progenitor cells. 13.The method of claim 12, wherein step (a) is effected by subjecting theundifferentiated ES cells to at least one condition being selected froma group consisting of avoiding aggregation of ES cells, growth oncollagen, cell seeding concentration between 2×10⁴ and 1×10⁵ cells/cm²and presence of differentiation medium.
 14. The method of claim 12,wherein said cells smaller than 50 μm are isolated by filtration,morphometry and/or densitometry.
 15. The method of claim 14, whereinsaid isolation by filtration is effected via a filter having a pore sizesmaller than 50 μm.
 16. The method of claim 11, wherein saidundifferentiated ES cells are human ES cells.
 17. The method of claim11, wherein said growth factor is selected from the group consisting ofvascular endothelial growth factor (VEGF), angiopoietin (Ang), plateletderived growth factor (PDGF), ephrin (Eph), fibroblast growth factor(FGF), tumor growth factor (TGF), placental growth factor (PlGF), tumorgrowth factor (TGF), cytokines, erythropoietin, thrombopoietin,transferrin, insulin, stem cell factor (SCF), Granulocytecolony-stimulating factor (G-CSF) and Granulocyte-macrophage colonystimulating factor (GM-CSF).
 18. A method of preparing vascular tissue,the method comprising: (a) obtaining a population of vasculogenicprogenitor cells; and (b) culturing said population of vasculogenicprogenitor cells in the presence of at least one vasculogenic and/orangiogenic growth factor, under conditions suitable for inducingvascular tissue differentiation.
 19. The method of claim 18, whereinstep (a) is effected by: (a) culturing individual undifferentiated EScells in a manner suitable for inducing differentiation of theundifferentiated ES cells into vasculogenic progenitor cells therebyobtaining a mixed population of cells; and (b) isolating cells smallerthan 50 μm from said mixed population of cells, said cells smaller than50 μm being vasculogenic progenitor cells.
 20. The method of claim 19,wherein step (a) is effected by subjecting the undifferentiated ES cellsto at least one condition being selected from a group consisting ofavoiding aggregation of ES cells, growth on collagen, cell seedingconcentration between 2×10⁴ and 1×10⁵ cells/cm² and presence ofdifferentiation medium.
 21. The method of claim 19, wherein said cellssmaller than 50 μm are isolated by filtration, morphometry and/ordensitometry.
 22. The method of claim 21, wherein said isolation byfiltration is effected via a filter having a pore size smaller than 50μm.
 23. The method of claim 19, wherein said undifferentiated ES cellsare human ES cells.
 24. The method of claim 18, wherein said populationof vasculogenic progenitor cells is cultured in a semi-solid,vascularization-promoting medium.
 25. The method of claim 18, whereinsaid population of vasculogenic progenitor is cultured on a3-dimensional scaffold.
 26. The method of claim 18, wherein saidvasculogenic and/or angiogenic factor is selected from the groupconsisting of vascular endothelial growth factor (VEGF), angiopoietin(Ang), platelet derived growth factor (PDGF), ephrin (Eph), fibroblastgrowth factor (FGF), tumor growth factor (TGF) and placental growthfactor (PlGF).
 27. A method of determining an effect of a factor onvascular development, growth and/or modification, the method comprising:(a) obtaining a population of vasculogenic progenitor cells; (b)exposing said population of vasculogenic progenitor cells to the factor;and (c) determining an effect of the factor on said population ofvasculogenic progenitor cells to thereby determine the effect thereof onvascular development.
 28. The method of claim 27, wherein step (a) iseffected by: (a) culturing individual undifferentiated ES cells in amanner suitable for inducing differentiation of the undifferentiated EScells into vasculogenic progenitor cells thereby obtaining a mixedpopulation of cells; and (b) isolating cells smaller than 50 μm fromsaid mixed population of cells, said cells smaller than 50 μm beingvasculogenic progenitor cells.
 29. The method of claim 28, wherein step(a) is effected by subjecting the undifferentiated ES cells to at leastone condition being selected from a group consisting of avoidingaggregation of ES cells, growth on collagen, cell seeding concentrationbetween 2×10⁴ and 1'10⁵ cells/cm² and presence of differentiationmedium.
 30. The method of claim 28, wherein said cells smaller than 50μm are isolated by filtration, morphometry and/or densitometry.
 31. Themethod of claim 30, wherein said isolation by filtration is effected viaa filter having a pore size smaller than 50 μm.
 32. The method of claim28, wherein said undifferentiated ES cells are human ES cells.
 33. Themethod of claim 27, wherein the factor is a substance and/or anenvironmental factor.
 34. The method of claim 27, wherein the factor isa putative angiogenesis and/or vasculogenesis downregulator, whereas themethod further comprising culturing said population of vasculogenicprogenitor cells under conditions suitable for promoting angiogenesisand/or vasculogenesis prior to step (b).
 35. The method of claim 27,wherein the factor is a putative angiogenesis and/or vasculogenesisupregulator, whereas the method further comprising culturing saidpopulation of vasculogenic progenitor cells under conditions limitingangiogenesis and/or vasculogenesis prior to step (b).
 36. A cell culturecomprising a population of vasculogenic progenitor cells beingsustainable in a proliferative state for at least 14 days and beingcapable of differentiation into smooth muscle, endothelial and/orhematopoietic cells upon exposure to at least one growth factor selectedfrom the group consisting of vascular endothelial growth factor (VEGF),angiopoietin (Ang), platelet derived growth factor (PDGF), ephrin (Eph),fibroblast growth factor (FGF), tumor growth factor (TGF), placentalgrowth factor (PIGF), cytokines, erythropoietin, thrombopoietin,transferrin, insulin, stem cell factor (SCF), Granulocytecolony-stimulating factor (G-CSF) and Granulocyte-macrophage colonystimulating factor (GM-CSF).
 37. The cell culture of claim 36, whereinsaid population of vasculogenic progenitor cells is capable ofexpressing at least one exogenous polypeptide.
 38. The cell culture ofclaim 37, wherein said exogenous polypeptide is selected from the groupconsisting of cell-surface markers, cell-surface antigens, angiogenicfactors, vasculogenic factors and hematopoietic factors.
 39. The cellculture of claim 37, wherein said exogenous polypeptide is expressed inan inducible manner.
 40. A composition of matter comprising a substrateand a population of vasculogenic progenitor cells, wherein saidvasculogenic progenitor cells are prepared from undifferentiated EScells by a method comprising the steps: (a) culturing individualundifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells thereby obtaining a mixed population of cells; and (b)isolating cells smaller than 50 μm from said mixed population of cells,said cells smaller than 50 μm being vasculogenic progenitor cells. 41.The composition of claim 40, wherein step (a) is effected by subjectingthe undifferentiated ES cells to at least one condition being selectedfrom a group consisting of avoiding aggregation of ES cells, growth oncollagen, cell seeding concentration between 2×10⁴ and 1×10⁵ cells/cm²and presence of differentiation medium.
 42. The composition of claim 41,wherein said cells smaller than 50 μm are isolated by filtration,morphometry and/or densitometry.
 43. The composition of claim 42,wherein said isolation by filtration is effected via a filter having apore size smaller than 50 μm.
 44. The composition of claim 40, whereinsaid undifferentiated ES cells are human ES cells.
 45. The compositionof claim 40, wherein said substrate is selected from the groupconsisting of matrigel, collagen gel, and polymeric scaffold.
 46. Thecomposition of claim 40, wherein said vasculogenic progenitor cellsbeing contacted with said substrate in a manner so as to induce vasculardevelopment within said substrate.
 47. A method of relieving orpreventing a vascular disease or condition in a mammalian subject, themethod comprising: (a) obtaining a population of vasculogenic progenitorcells; and (b) administering said vasculogenic progenitor cells into thesubject under conditions suitable for stimulating differentiation ofsaid vasculogenic progenitor cells into endothelial and smooth musclecells, thereby alleviating said vascular disease or condition.
 48. Themethod of claim 47, wherein obtaining said population of vasculogenicprogenitor cells is effected by: (a) culturing individualundifferentiated ES cells in a manner suitable for inducingdifferentiation of the undifferentiated ES cells into vasculogenicprogenitor cells; thereby obtaining a mixed population of cells; and (b)isolating cells smaller than 50 μm from said mixed population of cells,said cells smaller than 50 μm being vasculogenic progenitor cells. 49.The method of claim 48, wherein step (a) is effected by subjecting theundifferentiated ES cells to at least one condition being selected froma group consisting of avoiding aggregation of ES cells, growth oncollagen, cell seeding concentration between 2×10⁴ and 1×10⁵ cells/cm²and presence of differentiation medium.
 50. The method of claim 49,wherein said cells smaller than 50 μm are isolated by filtration,morphometry and/or densitometry.
 51. The method of claim 50, whereinsaid isolation by filtration is effected via a filter having a pore sizesmaller than 50 μm.
 52. The method of claim 47, wherein saidundifferentiated ES cells are human ES cells.
 53. The method of claim47, wherein said vascular disease or condition is selected from a groupconsisting of congenital vascular disorders, acquired vascular disordersand ischemia/reperfusion injury.
 54. A method of vascularizing amammalian tissue, the method comprising: (a) obtaining a population ofvasculogenic progenitor cells; (b) contacting said vasculogenicprogenitor cells with said mammalian tissue under conditions suitablefor stimulating differentiation of said vasculogenic progenitor cellsinto endothelial and smooth muscle cells, thereby enriching thevascularity of the tissue.
 55. The method of claim 54, wherein obtainingsaid population of vasculogenic progenitor cells is effected by: (a)culturing individual undifferentiated ES cells in a manner suitable forinducing differentiation of the undifferentiated ES cells intovasculogenic progenitor cells; thereby obtaining a mixed population ofcells; and (b) isolating cells smaller than 50 μm from said mixedpopulation of cells, said cells smaller than 50 μm being vasculogenicprogenitor cells.
 56. The method of claim 55, wherein step (a) iseffected by subjecting the undifferentiated ES cells to at least onecondition being selected from a group consisting of avoiding aggregationof ES cells, growth on collagen, cell seeding concentration between2×10⁴ and 1×10⁵ cells/Cm² and presence of differentiation medium. 57.The method of claim 56, wherein said cells smaller than 50 μm areisolated by filtration, morphometry and/or densitometry.
 58. The methodof claim 57, wherein said isolation by filtration is effected via afilter having a pore size smaller than 50 μm.
 59. The method of claim54, wherein said undifferentiated ES cells are human ES cells.
 60. Themethod of claim 54, wherein said mammalian tissue is an engineered,non-vascular tissue in need of vascularization.
 61. The method of claim54, wherein said mammalian tissue is an embryonic tissue.
 62. The methodof claim 54, wherein step (b) is performed in vitro.
 63. The method ofclaim 54, wherein step (b) is performed in vivo.
 64. A method ofrelieving or preventing a hematological disease or condition in amammalian subject, the method comprising: (a) obtaining a population ofvasculogenic progenitor cells; and (b) administering said vasculogenicprogenitor cells into the subject under conditions suitable forstimulating differentiation of said vasculogenic progenitor cells intoendothelial and blood cells, thereby alleviating said hematopoieticdisease or condition.
 65. The method of claim 64, wherein step (a) iseffected by: (a) culturing individual undifferentiated ES cells in amanner suitable for inducing differentiation of the undifferentiated EScells into vasculogenic progenitor cells, thereby obtaining a mixedpopulation of cells; and (b) isolating cells smaller than 50 μm fromsaid mixed population of cells, said cells smaller than 50 μm beingvasculogenic progenitor cells.
 66. The method of claim 65, wherein step(a) is effected by subjecting the undifferentiated ES cells to at leastone condition being selected from a group consisting of avoidingaggregation of ES cells, growth on collagen, cell seeding concentrationbetween 2×10⁴ and 1×10⁵ cells/cm² and presence of differentiationmedium.
 67. The method of claim 66, wherein said cells smaller than 50μm are isolated by filtration, morphometry and/or densitometry.
 68. Themethod of claim 67, wherein said isolation by filtration is effected viaa filter having a pore size smaller than 50 μm.
 69. The method of claim64, wherein said undifferentiated ES cells are human ES cells.
 70. Themethod of claim 64, wherein said hematological disease or condition isselected from a group consisting of congenital blood disorders, acquiredblood disorders, clotting disorders and neoplastic disease.
 71. A methodof preparing endothelial cells from vascular tissue, the methodcomprising: (a) subjecting the vascular tissue to conditions designedfor dissociating cells from the vascular tissue, thereby obtaining amixed population of dissociated cells; and (b) isolating cells smallerthan 50 μm from said mixed population of cells, said cells smaller than50 μm being endothelial cells.
 72. The method of claim 71, wherein saidcells smaller than 50 μm are isolated by filtration, morphometry and/ordensitometry.
 73. The method of claim 72, wherein said isolation byfiltration is effected via a filter having a pore size smaller than 50μm.
 74. The method of claim 71, wherein said vascular tissue is humanvascular tissue.
 75. A method of preparing epithelial cells fromvascular tissue, the method comprising: (a) subjecting the vasculartissue to conditions designed for dissociating cells from the vasculartissue, thereby obtaining a mixed population of dissociated cells,thereby obtaining a mixed population of individual cells; and (b)isolating cells larger than 50 μm from said mixed population of cells,said cells larger than 50 μm being epithelial cells.
 76. The method ofclaim 75, wherein said cells larger than 50 μm are isolated byfiltration, morphometry and/or densitometry.
 77. The method of claim 76,wherein said isolation by filtration is effected via a filter having apore size smaller than 50 μm.
 78. The method of claim 75, wherein saidvascular tissue is human vascular tissue.